Long-term

Results

of

a

Primary

End-to-end

Anastomosis

in

Peroperative

Detected

Bile

Duct

Injury

The

management

of

a

bile

duct

injury

detected

during

laparoscopic

cholecystectomy

is

still

under

discussion

An

end-to-end

anastomosis

-LRB-

with

or

without

T-tube

drainage

-RRB-

in

peroperative

detected

bile

duct

injury

has

been

reported

to

be

associated

with

stricture

formation

of

the

anastomosis

area

and

recurrent

jaundice

Between

and

of

a

total

of

bile

duct

injury

patients

were

referred

for

treating

complications

after

a

primary

end-to-end

anastomosis

After

referral

-LRB-

-RRB-

patients

were

initially

treated

endoscopically

or

by

percutaneous

transhepatic

stent

placement

-LRB-

n

-RRB-

After

a

mean

follow-up

of

years

patients

-LRB-

-RRB-

were

successfully

treated

with

dilatation

and

endoscopically

placed

stents

One

patient

died

due

to

a

treatment-related

complication

A

total

of

patients

-LRB-

-RRB-

underwent

a

hepaticojejunostomy

Postoperative

complications

occurred

in

three

patients

-LRB-

-RRB-

without

hospital

mortality

These

data

confirm

that

end-to-end

anastomosis

might

be

considered

as

a

primary

treatment

for

peroperative

detected

transection

of

the

bile

duct

without

extensive

tissue

loss

Complications

-LRB-

stricture

or

leakage

-RRB-

can

be

adequately

managed

by

endoscopic

or

percutaneous

drainage

the

majority

of

patients

-LRB-

-RRB-

and

reconstructive

surgery

after

complicated

end-to-end

anastomosis

is

a

procedure

with

relative

low

morbidity

and

no

mortality

Introduction

Bile

duct

injury

-LRB-

BDI

-RRB-

after

laparoscopic

cholecystectomy

-LRB-

LC

-RRB-

is

still

a

major

problem

in

current

surgical

practice

BDI

is

associated

with

reduced

survival

increased

morbidity

and

poor

long-term

quality

of

life

-LRB-

QoL

-RRB-

The

incidence

of

BDI

at

laparoscopic

cholecystectomy

has

been

reported

between

to

depending

on

the

criteria

used

to

define

the

injury

as

well

as

the

study

population

Of

these

injuries

one-third

is

detected

during

the

procedure

Measures

to

prevent

and

recognize

BDI

are

outlined

in

many

publications

The

optimal

treatment

strategy

and

short

and

long-term

outcome

has

been

published

extensively

Controversy

exists

however

about

the

management

of

peroperative

detected

BDI

The

most

important

factor

is

the

extent

of

tissue

loss

of

the

common

bile

duct

but

also

severity

of

inflammation

and

the

size

and

diameter

of

the

proximal

duct

The

peroperative

management

range

from

simple

drainage

and

referral

to

a

tertiary

center

to

an

end-to-end

anastomosis

-LRB-

EEA

-RRB-

-LRB-

duct

to

duct

with

or

without

T-tube

drainage

-RRB-

or

a

hepaticojejunostomy

-LRB-

HJ

-RRB-

It

has

been

suggested

that

EEA

is

associated

with

a

relative

high

stricture

rate

up

to

and

consequently

a

high

incidence

of

secondary

repair

Therefore

many

tertiary

centers

prefer

to

perform

a

HJ

instantly

A

secondary

repair

after

EEA

should

be

associated

with

an

increased

risk

of

postoperative

complications

as

the

formation

for

strictures

and

stenosis

Others

however

consider

EEA

as

a

relative

simple

definitive

repair

and

also

an

optimal

initial

drainage

procedure

before

reconstructive

surgery

in

a

secondary

setting

Reports

on

large

consecutive

series

to

analyze

the

outcome

of

EEA

are

scary

because

this

procedure

is

generally

not

performed

in

referral

centers

One

should

realize

that

patients

referred

to

such

a

center

after

previous

EEA

elsewhere

are

a

negative

selection

of

the

EEA

population

So

far

a

systematic

analysis

of

a

large

group

of

patients

with

an

EEA

has

not

been

performed

and

therefore

this

study

was

conducted

The

aim

of

the

present

study

was

to

analyze

short

and

long-term

outcome

in

patients

who

are

referred

after

failure

of

a

primary

EEA

Patients

and

Methods

Patients

Cohort

and

Data

Collection

Between

January

and

January

consecutive

patients

were

referred

to

the

Academic

Medical

Center

-LRB-

AMC

-RRB-

in

Amsterdam

for

the

management

of

a

BDI

after

cholecystectomy

Patient

data

was

induced

in

a

prospective

database

All

types

of

BDI

were

included

also

minor

injuries

such

as

leakage

from

the

cystic

duct

or

ducts

of

Luschka

To

define

the

location

of

BDI

the

Bismuth

classification

was

used

For

the

present

study

the

medical

charts

of

all

patients

who

underwent

a

primary

EEA

were

retrospectively

reviewed

to

analyze

the

initial

operation

reports

and

clinical

data

Data

from

the

referring

hospital

included

indication

for

cholecystectomy

type

of

initial

procedure

location

of

injury

type

of

repair

including

the

use

of

a

T-tube

the

postoperative

diagnostic

interventions

and

the

therapeutic

interventions

before

referral

Data

from

the

present

center

included

symptoms

at

referral

diagnostic

work-up

type

of

treatment

short-term

and

long-term

complications

Endoscopic

Radiological

and

Surgical

Treatment

for

complicated

EEA

Endoscopic

treatment

was

performed

by

balloon

dilatation

or

catheter

dilatation

before

stent

placement

The

biliary

stent

is

placed

over

the

guide

wire

bridging

the

stenosis

Two

or

more

stents

were

inserted

if

possible

For

multiple

stent

insertion

an

endoscopic

sphincterotomy

was

performed

to

facilitate

stent

placement

Stents

were

replaced

after

weeks

and

subsequently

exchanged

every

months

to

avoid

cholangitis

Percutaneous

transhepatic

catheterization

was

performed

by

injecting

the

contrast

medium

from

the

right

intercostal

approach

A

right

or

left

approach

for

the

percutaneous

transhepatic

biliary

drainage

was

chosen

depending

on

ultrasound

images

illustrating

the

biliary

anatomy

and

the

possibility

of

puncturing

a

dilated

intrahepatic

bile

duct

Catheterization

of

intrahepatic

bile

ducts

was

performed

in

standard

fashion

A

guide

wire

was

advanced

through

the

biliary

stricture

into

the

duodenum

When

this

was

achieved

a

biliary

drainage

catheter

was

inserted

All

drainage

procedures

were

performed

with

the

administration

of

broad-spectrum

antibiotics

In

case

of

a

surgical

reconstruction

the

procedure

was

performed

via

a

Roux-en-Y

hepaticojejunostomy

The

stricture

in

the

CBD

is

transected

and

the

hilar

plate

is

opened

The

hepatic

ducts

of

different

segmental

bile

ducts

are

mobilized

and

from

there

opened

over

the

left

hepatic

duct

Intrahepatic

segmental

ducts

are

mobilized

and

if

possible

sutured

together

before

one

or

two

jejunal

anastomosis

are

made

A

closed

suction

drain

is

placed

during

operation

and

removed

hours

after

surgery

Percutaneous

transhepatic

drains

when

inserted

before

surgery

are

left

in

place

and

removed

after

days

till

weeks

depending

on

the

clinical

course

the

level

of

anastomosis

and

the

surgeons

preference

Outcome

Follow-up

data

was

obtained

through

outpatient

records

and

the

records

of

the

general

practitioner

The

outcome

of

treatment

was

analyzed

by

the

number

complications

and

late

restenosis

during

follow-up

Failure

of

treatment

was

defined

as

recurrent

stenosis

after

stent

therapy

followed

by

surgery

or

recurrent

stenosis

after

surgical

reconstruction

followed

by

additional

therapy

Statistical

Analysis

Data

from

patient

characteristics

management

and

outcome

show

descriptive

statistics

in

number

of

patients

and

percentages

Mean

and

median

values

are

given

with

a

minimum

and

maximum

Long-term

stricture-free

survival

was

analyzed

by

Kaplan

Meier

Survival

Analysis

Data

analyses

were

performed

using

SPSS

software

-LRB-

SPSS

Chicago

Illinois

USA

-RRB-

Results

Patients

Characteristics

at

Referral

The

referral

pattern

of

BDI

patients

-LRB-

n

-RRB-

throughout

the

last

years

are

summarized

in

Fig.

From

the

total

of

patients

-LRB-

-RRB-

underwent

a

primary

EEA

Patient

characteristics

are

listed

in

Table

The

laparoscopic

cholecystectomy

-LRB-

n

-RRB-

was

converted

in

all

patients

An

open

cholecystectomy

was

performed

in

eight

patients

-LRB-

-RRB-

In

patients

-LRB-

-RRB-

the

anastomosis

was

made

over

a

T-tube

The

tube

was

removed

at

the

referring

hospital

or

at

the

AMC

after

a

mean

of

days

-LRB-

range

days

-RRB-

Figure

patients

for

treatment

of

bile

duct

injury

Total

number

of

referred

patients

-LRB-

red

-RRB-

patients

referred

after

a

primary

end

to

end

anastomosis

-LRB-

blue

-RRB-

and

patients

referred

after

a

primary

biliodigestive

reconstruction

-LRB-

green

-RRB-

Table

Characteristics

Primary

EEAn

Age

at

cholecystectomyMean

-LRB-

years

-RRB-

for

cholecystectomySymptomatic

a

of

initial

operationOpen

to

open

over

of

T-tube

in

situDays

median

-LRB-

range

-RRB-

-LRB-

-RRB-

After

the

primary

EEA

patients

-LRB-

-RRB-

underwent

other

therapeutical

interventions

before

referral

-LRB-

Table

-RRB-

-LRB-

Fig.

-RRB-

These

patients

underwent

a

range

of

one

to

three

procedures

before

referral

-LRB-

median

-RRB-

The

interventions

included

a

relaparotomy

in

two

patients

-LRB-

-RRB-

percutaneous

drainage

of

fluid

collections

in

five

patients

-LRB-

-RRB-

endoscopicially

placed

stents

in

patients

-LRB-

-RRB-

a

papillotomy

in

nine

patients

-LRB-

-RRB-

and

percutaneous

transhepatic

drainage

in

two

patients

-LRB-

-RRB-

The

median

interval

from

the

primary

EEA

to

referral

was

weeks

-LRB-

range

weeks

-RRB-

At

referral

a

biliary

stricture

was

diagnosed

in

patients

-LRB-

-RRB-

in

patients

-LRB-

-RRB-

bile

leakage

was

diagnosed

and

combination

of

both

in

eight

patients

-LRB-

-RRB-

Symptoms

at

referral

were

cholestasis

-LRB-

n

-RRB-

cholangitis

-LRB-

n

-RRB-

and

abdominal

pain

-LRB-

n

-RRB-

Three

patients

were

referred

because

of

uncontrolled

sepsis

-LRB-

n

-RRB-

and

peritonitis

-LRB-

n

-RRB-

According

to

the

Bismuth

classification

the

majority

of

injuries

-LRB-

leakage

of

stricture

-RRB-

-LRB-

n

-RRB-

was

located

below

the

bifurcation

In

nine

patients

-LRB-

-RRB-

the

injury

-LRB-

mostly

strictures

-RRB-

involved

the

bifurcation

or

the

right

or

left

hepatic

duct

-LRB-

i.e.

Bismuth

classification

grades

IV

and

V

-RRB-

Table

Pattern

Primary

EEAn

Time

interval

between

injury

and

referral

Weeks

median

-LRB-

range

-RRB-

-LRB-

-RRB-

Intervention

after

EEA

and

before

referral

Explorative

Percutaneous

Endoscopic

Endoscopic

at

referral

Abdominal

Uncontrolled

at

of

stenosis

and

of

injury

at

referralb

transhepatic

drainagebAccording

to

Bismuth

classificationFigure

showing

successful

-LRB-

aggressive

-RRB-

stent

therapy

after

primary

EEA

a

Stenosis

of

the

common

bile

duct

b

Stents

in

situ

c

After

stent

removal

within

a

year

Management

after

Referral

Diagnostic

work-up

was

performed

by

CT-scan

-LRB-

n

-RRB-

endoscopic

cholangiography

-LRB-

n

-RRB-

and

transhepatic

cholangiography

-LRB-

n

-RRB-

The

definitive

treatment

of

BDI

patients

after

EEA

is

shown

in

the

flow

diagram

-LRB-

Fig.

-RRB-

After

work-up

three

patients

-LRB-

-RRB-

were

treated

with

percutaneous

transhepatic

cholangiographic

drainage

-LRB-

PTCD

-RRB-

and

patients

-LRB-

-RRB-

were

treated

endoscopically

Thirteen

patients

-LRB-

-RRB-

underwent

reconstructive

surgery

after

work-up

eight

patients

because

of

a

complete

stenosis

of

the

CBD

in

three

patients

reconstructive

surgery

was

performed

after

failure

of

stent

therapy

at

the

referring

hospital

and

in

two

patients

because

of

a

percutaneous

fistula

and

persistent

bile

leakage

Figure

diagram

of

the

success

and

failure

rates

after

a

multidisciplinary

treatment

of

patients

who

underwent

a

peroperative

end

to

end

anastomosis

for

bile

duct

injury

Given

percentages

are

calculated

from

the

number

of

patients

in

the

previous

flow

box

PTCD

Percutaneous

transhepatic

catheter

dilatation

Radiological

and

Endoscopic

Treatment

Three

patients

were

successfully

treated

by

PTCD

In

two

patients

a

stenosis

was

treated

by

transhepatic

dilatation

and

in

one

patient

bile

leakage

was

treated

by

external

transhepatic

stent

insertion

Forty

patients

-LRB-

-RRB-

were

treated

endoscopically

-LRB-

Fig.

-RRB-

In

patients

-LRB-

-RRB-

stent

insertion

was

successful

and

in

three

patients

-LRB-

-RRB-

adequate

drainage

succeeded

by

papillotomy

The

median

number

of

stent

replacements

was

five

-LRB-

range

-RRB-

with

a

median

duration

of

treatment

of

days

-LRB-

range

-RRB-

Complications

occurred

in

nine

patients

-LRB-

-RRB-

Stent

dislodgment

-LRB-

n

-RRB-

clogging

-LRB-

n

-RRB-

and

cholangitis

-LRB-

n

-RRB-

were

mild

complications

and

were

successfully

treated

by

stent

exchange

or

administration

of

antibiotics

One

severe

complication

occurred

in

a

patient

After

years

of

stent

therapy

the

stent

migrated

and

perforated

the

duodenum

Finally

the

patient

died

due

to

multiple

organ

failure

and

sepsis

Surgical

Treatment

After

referral

and

during

the

follow-up

period

a

new

hepatobiliary

anastomosis

was

performed

by

hepaticojejunostomy

in

patients

-LRB-

-RRB-

Mean

duration

of

hospital

stay

was

days

Postoperative

complications

occurred

in

one

patient

-LRB-

-RRB-

who

underwent

a

PTC

procedure

after

leakage

of

the

anastomosis

No

hospital

mortality

occurred

in

patients

who

underwent

a

reconstructive

procedure

after

a

previous

EEA

Long-term

Follow-up

After

a

mean

follow-up

of

years

seven

patients

-LRB-

-RRB-

have

died

One

endoscopically

treated

patient

died

due

to

a

complication

of

treatment

as

described

above

The

other

patients

died

due

to

malignancy

-LRB-

n

-RRB-

and

myocardial

infarct

-LRB-

n

-RRB-

The

long-term

results

in

patients

treated

with

endoscopic

and

radiological

treatment

are

as

follows

from

a

total

of

patients

treated

with

endoscopic

or

PTCD

procedures

-LRB-

n

-RRB-

was

successful

In

three

patients

-LRB-

-RRB-

signs

of

restenosis

occurred

after

stent

removal

after

and

months

Continued

stent

therapy

was

successful

in

all

three

patients

Five

patients

underwent

reconstructive

surgery

after

prolonged

endoscopic

stenting

Postoperative

complications

occurred

in

two

of

the

five

patients

and

these

patients

received

additional

therapy

for

wound

infection

-LRB-

n

-RRB-

and

postoperative

cholangitis

-LRB-

n

-RRB-

The

long-term

results

of

surgical

treatment

after

EEA

are

as

follows

from

patients

who

underwent

a

HJ

after

work-up

a

stenosis

of

the

anastomosis

occurred

in

two

patients

-LRB-

-RRB-

Both

patients

underwent

successful

percutaneous

transhepatic

dilatation

respectively

and

months

after

surgery

The

overall

years

stricture

free

survival

in

the

total

cohort

-LRB-

n

-RRB-

is

shown

by

a

Kaplan

Meier

curve

in

Fig.

Figure

Meier

plot

showing

proportion

of

patients

without

restenosis

among

bile

duct

injury

patients

treated

for

complications

after

EEA

Discussion

The

present

study

describes

a

selected

group

of

BDI

patients

who

were

referred

for

treatment

after

a

complicated

EEA

This

group

of

patients

is

a

negative

selection

representing

the

worst

complications

of

EEA

otherwise

patients

were

not

referred

for

additional

treatment

So

this

study

does

not

provide

any

information

about

the

success

rate

of

EEA

The

present

study

shows

a

long-term

stricture

free

survival

of

in

EEA

patients

after

treatment

in

a

tertiary

center

The

analysis

showed

that

even

the

majority

of

complications

after

primary

AEE

in

a

general

hospital

can

successfully

be

treated

by

endoscopic

and

radiological

interventions

In

only

one-third

of

the

patients

a

secondary

surgical

repair

is

necessary

The

surgical

reconstruction

after

EEA

was

associated

with

acceptable

morbidity

and

without

mortality

Around

to

patients

are

referred

annually

without

any

sign

of

decrease

over

the

last

years

Considering

LC

's

per

year

in

the

Netherlands

we

still

consider

mentioned

in

the

reviews

as

an

underestimation

of

the

real

incidence

of

BDI

at

least

in

the

Netherlands

In

of

the

patients

referred

to

the

AMC

the

injury

was

detected

during

the

initial

surgical

procedure

This

finding

is

similar

to

reports

in

literature

From

the

total

of

referred

BDI

patients

was

referred

for

the

treatment

of

a

complication

after

peroperative

EEA

Because

referred

patients

only

represent

the

complications

after

EEA

we

do

not

know

the

real

incidence

of

EEA

procedures

in

BDI

Peroperative

repair

in

BDI

detected

during

surgery

can

be

performed

by

EEA

-LRB-

with

or

without

the

use

of

a

T-tube

-RRB-

or

by

a

primary

HJ

A

HJ

is

a

more

complex

procedure

and

one

should

be

very

careful

not

to

further

extend

the

injury

into

the

intrahepatic

ducts

or

subsequently

damage

the

arterial

supply

-LRB-

bleeding

and

clipping

or

ligation

of

right

hepatic

artery

-RRB-

The

present

study

shows

that

if

complications

occur

after

EEA

these

can

successfully

be

treated

by

percutaneous

or

endoscopic

balloon

dilatation

and/or

stenting

in

the

majority

of

patients

A

HJ

in

the

acute

setting

without

dilated

bile

ducts

is

even

more

difficult

and

therefore

consulting

a

surgeon

with

experience

in

reconstructive

hepatobiliary

surgery

is

recommended

In

contrast

with

a

primary

HJ

an

EEA

is

a

relatively

simple

procedure

and

can

also

be

performed

in

less

experienced

hands

The

risk

to

increase

damage

is

smaller

in

an

EEA

procedure

and

with

the

use

of

a

T

tube

instant

bile

drainage

is

realized

If

indicated

reconstructive

surgery

by

means

of

an

elective

HJ

can

be

performed

It

is

strongly

advised

to

perform

a

HJ

after

classification

the

injury

and

analyzing

the

biliary

anatomy

Preoperative

cholangiography

-LRB-

with

the

use

of

the

T

tube

-RRB-

will

illustrate

the

location

of

the

stenosis

and

the

extension

of

dilation

of

the

proximal

bile

ducts

A

reconstructive

procedure

for

stenosis

of

EEA

has

a

satisfying

outcome

as

peroperative

conditions

are

good

after

the

inflammation

has

subsided

and

the

bile

ducts

are

dilated

due

to

stenosis

In

a

situation

in

which

peroperative

bile

leakage

is

due

to

-LRB-

extensive

-RRB-

tissue

loss

in

particular

in

patients

with

more

proximal

lesions

at

the

bifurcation

or

intrahepatically

no

primary

repair

should

be

performed

In

this

situation

adequate

drainage

of

the

upper

right

abdomen

is

strongly

advised

and

the

patient

should

be

referred

for

elective

reconstruction

Referral

to

tertiary

center

in

this

situation

has

a

positive

effect

on

outcome

End-to-end

anastomosis

is

reported

to

be

associated

with

a

high

incidence

of

recurrent

jaundice

due

to

stricture

formation

of

the

anastomotic

area

Therefore

some

authors

suggest

that

EEA

is

almost

never

appropriate

if

the

bile

duct

has

been

completely

transected

while

others

favor

this

strategy

when

there

is

no

extensive

tissue

loss

Stent

therapy

for

iatrogenic

bile

duct

strictures

has

changed

during

the

last

decade

and

therefore

the

long-term

outcome

after

stenting

has

improved

A

more

aggressive

approach

with

more

stents

and

smaller

time

intervals

between

stent

changes

is

favored

With

this

new

approach

of

the

patients

who

undergo

an

ERCP

for

postoperative

bile

duct

stenosis

have

a

stricture-free

survival

Although

complications

occur

at

a

significant

rate

these

are

usually

mild

The

only

severe

complication

occurred

in

the

present

series

due

to

a

migrated

stent

was

not

reported

in

previous

series

After

stent

removal

recurrent

stenosis

develops

in

of

patients

within

years

of

stent

removal

Therefore

endoscopic

treatment

should

be

the

initial

management

of

choice

for

postoperative

bile

duct

strictures

Without

signs

of

improvement

after

endoscopic

stenting

reconstructive

surgery

is

indicated

in

otherwise

fit

patients

Of

interest

is

the

evaluation

of

the

long-term

stricture-free

survival

after

treatment

for

complications

after

EEA

After

a

mean

follow-up

of

years

restenosis

after

treatment

developed

in

of

the

patients

In

all

patients

who

underwent

initial

endoscopic

therapy

restenosis

occurred

a

relatively

short

time

after

stent

removal

diagnosed

within

to

months

follow-up

Therefore

endoscopic

treatment

is

not

associated

with

a

high

rate

of

long-term

restenosis

after

stent

removal

In

two

patients

a

restenosis

occurred

within

years

after

a

hepaticojejunostomy

Symptoms

were

cholestasis

and

cholangitis

In

both

patients

transhepatic

dilatation

was

successful

to

resolve

the

stenosis

The

long-term

stricture-free

survival

of

in

the

present

series

provides

evidence

for

a

good

outcome

after

treating

complicated

EEA

patients

If

BDI

is

detected

during

surgery

in

particular

if

there

is

no

extensive

tissue

loss

the

local

anatomy

is

clear

and

there

is

no

inflammation

EEA

could

be

considered

as

a

sufficient

treatment

strategy

Patients

with

postoperative

complications

-LRB-

stricture

or

leakage

-RRB-

should

be

treated

by

a

multidisciplinary

team

of

gastroenterologists

radiologists

and

surgeons

Postoperative

complications

can

adequately

be

managed

by

endoscopic

or

percutaneous

drainage

in

two-third

of

the

patients

Reconstructive

surgery

after

a

complicated

EEA

is

associated

with

low

morbidity

and

no

mortality

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


Conduction velocities in amphibian skeletal muscle fibres exposed to hyperosmotic extracellular solutions Early quantitative analyses of conduction velocities in unmyelinated nerve studied in a constantly iso-osmotic volume conductor were extended to an analysis of the effects of varying extracellular osmolarities on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle fibres . Previous papers had reported that skeletal muscle fibres exposed to a wide range of extracellular sucrose concentrations resemble perfect osmometers with increased extracellular osmolarity proportionally decreasing fibre volume and therefore diminishing fibre radius , a. However , classical electrolyte theory -LRB- Robinson and Stokes 1959 , Electrolyte solutions 2nd edn . Butterworth & Co. pp 41 -- 42 -RRB- would then predict that the consequent increases in intracellular ionic strength would correspondingly decrease sarcoplasmic resistivity , Ri . An extension of the original cable analysis then demonstrated that the latter would precisely offset its expected effect of alterations in a on the fibre axial resistance , ri , and leave action potential conduction velocity constant . In contrast , other reports -LRB- Hodgkin and Nakajima J Physiol 221:105 -- 120 , 1972 -RRB- had suggested that Riincreased with extracellular osmolarity , owing to alterations in cytosolic viscosity . This led to a prediction of a decreased conduction velocity . These opposing hypotheses were then tested in muscle fibres subject to just-suprathreshold stimulation at a Vaseline seal at one end and measuring action potentials and their first order derivatives , dV/dt , using 5 -- 20 MΩ , 3 M KCl glass microelectrodes at defined distances away from the stimulus sites . Exposures to hyperosmotic , sucrose-containing , Ringer solutions then reversibly reduced both conduction velocity and maximum values of dV/dt . This was compatible with an increase in Ri in the event that conduction depended upon a discharge of membrane capacitance by propagating local circuit currents through initially passive electrical elements . Conduction velocity then showed graded decreases with increasing extracellular osmolarity from 250 -- 750 mOsm . Action potential waveforms through these osmolarity changes remained similar , including both early surface and the late after-depolarisation events reflecting transverse tubular activation . Quantitative comparisons of reduced-χ 2 values derived from a comparison of these results and the differing predictions from the two hypotheses strongly favoured the hypothesis in which Riincreased rather than decreased with hyperosmolarity . Introduction A classic paper by Hodgkin -LRB- 1954 -RRB- -LRB- see also : Adrian 1975 ; Noble 1979 -RRB- performed a quantitative cable analysis of the local circuit currents thought responsible for action potential propagation in unmyelinated nerve fibres studied in large volumes of constantly iso-osmotic extracellular fluid and demonstrated that their conduction velocities should vary as the square root of fibre radius , a. Action potential conduction velocity is also physiologically important in striated muscle : it ensures a rapid , sarcomeric activation leading to near-synchronous muscle contraction . The present paper accordingly extends these early analyses to effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle . Skeletal muscle membrane differs from unmyelinated nerve in including an excitable transverse -LRB- T - -RRB- system whose activation initiates muscle contraction -LRB- Adrian et al. 1969 , 1970 ; Huang 1993 ; Nielsen et al. 2004 ; Stephenson 2006 -RRB- . Nevertheless , it is likely that the initial sarcolemmal membrane activation precedes the subsequent inward tubular excitation and that the latter may take place partially independently of the propagation of the surface excitation wave . Sheikh et al. -LRB- 2001 -RRB- suggested a partially separable T-tubule excitation initiated by Na + channels selectively clustered around the mouths of the T-tubular lumina : detubulation produced by osmotic shock left surface membrane conduction velocities unchanged . Furthermore , increases in extracellular osmolarity did not alter the tubular diameters at the necks of the T-tubular system important in electrical connectivity between T-tubule network and remaining extracellular space -LRB- Launikonis and Stephenson 2002 , 2004 -RRB- . However , variations in surface conduction velocities in skeletal muscle studied under varying osmolarities would be expected to differ from those of nerve fibres studied under constant , iso-osmotic conditions owing to the consequent changes in their cell volumes and fibre diameters . Nevertheless , skeletal muscle volume exhibits close to ideal osmotic behaviour , varying inversely with changes in extracellular osmolarity that would in turn predictably increase solute concentration . The Results section of this paper accordingly first develops the original treatment -LRB- Hodgkin 1954 ; Adrian 1975 ; Noble 1979 -RRB- for situations in which fibre diameter is varied by alterations in extracellular osmolarity that would in turn alter intracellular ionic strength . This provided quantitative expectations for any resulting change in conduction velocity that corresponded to two contrasting situations . First , classical electrolyte theory -LRB- Robinson and Stokes 1959 ; Atkins 1998 -RRB- would predict that increased intracellular ionic strength should proportionally decrease sarcoplasmic resistivity Ri . Our analysis then indicated that this would precisely offset any expected changes in fibre axial resistance , ri , produced by the osmotically induced alterations in fibre diameter and thereby leave conduction velocity unchanged . Secondly , Hodgkin and Nakajima -LRB- 1972 -RRB- reported that Ri increased with extracellular osmolarity , possibly due to increases in myoplasmic viscosity , although we certainly do not exclude possible factors arising from the more complex membrane structures found in muscle as opposed to nerve -LRB- see e.g. Sheikh et al. 2001 -RRB- . This led to predictions that conduction velocity would decrease with increasing extracellular osmolarity . These predictions were then investigated by experimental determinations of conduction velocity obtained from action potential records derived from microelectrode measurements made at known distances from defined stimulation sites in surface muscle fibres of Rana esculenta studied at a fixed -LRB- ∼ 7 °C -RRB- temperature at varied extracellular osmolarities . These findings demonstrated reversible changes in the conduction velocities of action potentials the nature of whose waveforms , including early surface and later tubular components , were otherwise unchanged , and distinguished between hypotheses through the observed dependence of conduction velocity on extracellular osmolarity . Materials and methods Cold-adapted frogs , Rana esculenta , were killed by concussion followed by pithing -LRB- Schedule I : Animal Procedures Act , Home Office , UK -RRB- . The skin was removed and the tendon of insertion of the sartorius into the patella ligated , cut distally and dissected along its borders up to and including its origin at the pelvic girdle and acetabulum . This was performed at room temperature -LRB- 21.7 ± 0.51 °C ; mean ± standard error of the mean ; n = 15 -RRB- in standard Ringer solution consisting of -LRB- mM -RRB- : 115 NaCl , 2.5 KCl , 1.8 CaCl2 , 3 Hepes , titrated to pH 7 , osmolarity 250 mOsm . The muscle was then stretched to 1.4 times its in situ length and secured in a transilluminated methacrylate polymer -LRB- Perspex -RRB- chamber by pinning the cleaned acetabulum and the ligature to minimize contractile artefact , which became less evident in solutions of higher osmolarities , and entirely absent with the higher -LRB- > 350 − 400 mM -RRB- sucrose concentrations . The isolated sartorius muscles had an in situ length of 30.6 ± 0.94 mm and a length of 41.6 ± 1.15 mm -LRB- n = 15 -RRB- when stretched . The muscle was laid over a supporting ramp with its dorsal surface uppermost . A watertight Perspex partition , coated with a layer of Vaseline , with the muscle running through a notch at its lower surface electrically isolated the recording chamber into two compartments . The only path conducting the brief voltage stimuli applied from two platinum electrodes at opposite ends of the chamber was therefore through the muscle across the partition . The platinum electrode on the side of the chamber containing the acetabulum was the cathode . The temperature of the bath solution was lowered by circulating distilled water cooled in a magnetically stirred ice bath . The water was circulated through a glass coil placed in the chamber in close proximity to the muscle , using a Minipuls 3 peristaltic pump -LRB- Gilson , France -RRB- . A digital thermometer incorporating a remote thermistor -LRB- P. Frost , Department of Physiology , Development and Neuroscience , Cambridge -RRB- , previously linearised and calibrated against a platinum film resistor was placed in the bathing solution near the muscle to allow the temperature to be continuously monitored and adjusted to remain constant throughout the recordings . The temperature was kept constant at 7.0 ± 0.03 °C -LRB- n = 15 -RRB- -LRB- Sheikh et al. 2001 -RRB- . Action potentials are temperature-sensitive so this allowed conditions to be optimised . Cooling also prolonged fibre viability especially in the hyperosmotic conditions . The muscle was studied in the range of extracellular osmolarities from 250 to 750 mOsm . Solutions of different osmolarities were made by adding to the standard Ringer solution the membrane-impermeant solute sucrose at varying -LRB- 150 , 250 , 350 , 400 , 450 , 500 mM respectively -RRB- concentrations to yield solutions whose increased osmolarities -LRB- mOsm -RRB- were calculated from their total solute concentrations , and checked against measurements using a standard vapour pressure osmometer . Hepes was obtained from Sigma-Aldrich -LRB- Gillingham , Dorset , UK -RRB- and all other reagents from BDH Limited -LRB- UK -RRB- . Solutions were changed , avoiding contact with the muscle , between tests using a vacuum pump -LRB- Edwards , UK -RRB- after which approximately 5 -- 10 min elapsed before recording to permit time for both temperature -LRB- 7 °C -RRB- and osmotic equilibration ; previous reports -LRB- Ferenczi et al. 2004 -RRB- have indicated that 200 s sufficed to permit complete volume changes in response to hyperosmotic solutions . 3 M KCl-filled glass capillary microelectrodes -LRB- resistances 5 -- 20 MΩ ; tip potentials < 5 mV : Adrian 1956 -RRB- drawn from 1.2 mm -LRB- inner diameter -RRB- glass tubing were used to obtain membrane potentials . These were mounted via Ag/AgCl junctions to a high impedance voltage follower balanced by matching Ag/AgCl junctions earthing the bath . Only surface fibres showing clear-cut penetrations and stable resting membrane potentials were studied . Rates of rise and fall , dV/dt , in the voltage traces , were obtained by electrical differentiation of the output , with voltage and dV/dt channels filtered between cutoffs of 0 Hz/5 kHz and 0 Hz/20 kHz respectively . Action potentials were elicited by direct just-suprathreshold stimulation which varied between 1.5 and 5 V , to minimise electrotonic spread of the stimulus voltage and the number of fibres in which electrical activity was initiated , across the Perspex partition , through two platinum stimulating electrodes . Conduction velocity was calculated by dividing the distance from the site of action potential initiation to the position of the recording microelectrode tip , ascertained by a Vernier scale , which gave measurements in cm to two decimal places , by the latency given by the time from a clear stimulus artefact generated to the peak of the dV/dt input , which will be referred to as the maximum dV/dt . The corresponding resting membrane potential , action potential overshoot and the value of maximum dV/dt were also noted . These relatively early events in the timecourse of the regenerative response often could be measured even in records showing small contractile artefact , which often only became evident at later times in the recorded traces . Results The experiments utilized an extension of early quantitative analyses -LRB- Hodgkin 1954 ; Adrian 1975 -RRB- of conduction velocity in unmyelinated nerve studied in a constantly iso-osmotic volume conductor to changes that would result from varying the extracellular osmolarities in Rana esculenta skeletal muscle fibres . The initial analysis used the cable equation for the current density through any patch of flat membrane , expressed as current per unit membrane area , This describes conduction in a cable whose own finite internal volume is small compared to that of the extracellular conducting fluid in which it is studied to give large ratios between longitudinal intracellular , ri , and extracellular , ro , resistances to current flow per unit length , i.e. , ro << ri , permitting extracellular terms relating to ro to be dropped . Equation -LRB- 1 -RRB- is combined with the expression for conduction velocity , using the chain rule of differentiation , to give : where Vm is the internal potential across the membrane at distance x along the fibre . Conduction at constant velocity through local circuit spread of excitation then requires Im to be a single valued function of Vm . The term containing the intrinsic membrane parameters consisting of the area of membrane per unit length of fibre s , and the axial cytoplasmic resistance per unit length ri , is then constant , whence , and where the constant k only depends on passive local membrane electrical properties . Thus : For unmyelinated cylindrical fibres for which the axial cytoplasmic resistance per unit length , ri , is related to fibre radius a and sarcoplasmic resistivity Ri by Ri = riπ a2 , the total fibre volume , vol , = π a2L where L is the length of the fibre and for which the total surface area of the fibre , A = sL : In the case of skeletal muscle fibres exposed to solutions of different osmolarities , osm , the volume , vol , behaves as a perfect osmometer following the relationship : and reductions in fibre volume due to increased extracellular osmolarity would correspondingly increase solute concentration . Classical electrolyte theory -LRB- Robinson and Stokes 1959 -RRB- then predicts that each participating ion contributes a specific conductance Ksp determined by its solute concentration ηC and a constant Λ defined for any given ion in any specified solute and referred to the conductivity of the ion at a 1 M concentration : The overall conductivity of such an ideal solution is then the algebraic sum of the conductivities of individual component ions . Decreases in fibre volume resulting from increased extracellular osmolarities then would increase intracellular ionic strength and in turn increase Ksp and proportionally decrease Ri : Equation -LRB- 7 -RRB- then gives : Equations -LRB- 5 -RRB- and -LRB- 8 -RRB- then give , thereby predicting a constant conduction velocity in view of the fact that the terms k ′ , resulting from the additional proportionalities above , and A are both constants . In contrast , Hodgkin and Nakajima -LRB- 1972 -RRB- suggested that sarcoplasmic resistivity , Ri , increased with increasing extracellular osmolarity , possibly owing to an increased myoplasmic viscosity , with reductions in fibre volume in hyperosmotic solutions , reporting a linear increase in Ri with increasing extracellular osmolarity : where D and E are constants . From -LRB- 5 -RRB- and -LRB- 10 -RRB- : Because k ′ and A are constants , we have : where D ′ and E ′ are also constants . This forms a contrasting expectation in which θ is expected to decrease with increasing extracellular osmolarity in a relationship modelled by Eq . -LRB- 12 -RRB- as a result of an increase in Ri . The alternative hypotheses could then be quantitatively tested by measurements of θ under different conditions of extracellular osmolarity . Reversible effects of hyperosmotic extracellular solutions on action potential waveforms and latencies Figure 1 shows typical action potential and dV/dt waveforms obtained at a temperature of ∼ 7 °C before , during and after exposures to solutions with increased extracellular osmolarity with column A showing the action potential waveforms and column B their corresponding dV/dt records . Panel a shows typical results from fibres in iso-osmotic Ringer at 7 °C . The muscle was then exposed to a hyperosmotic -LRB- 600 mOsm -RRB- Ringer solution and recordings resumed 5 -- 10 min following this solution change -LRB- Panel b -RRB- . Finally , the muscle was then returned to iso-osmotic Ringer and recordings again resumed 5 -- 10 min after the solution was restored -LRB- Panel c -RRB- . Latencies were measured from the clear stimulus artefact to the maximum point of the dV/dt trace as seen in column B : this approach provided more consistent measurements of action potential latency than using arbitrarily chosen points on the action potential traces -LRB- see also Sheikh et al. 2001 -RRB- . Fig. 1Typical action potential -LRB- A -RRB- and dV/dt traces -LRB- B -RRB- obtained from muscle fibres exposed successively to -LRB- a -RRB- iso-osmotic Ringer -LRB- b -RRB- hyperosmotic -LRB- 600 mOsm -RRB- Ringer and -LRB- c -RRB- returned to iso-osmotic Ringer , in the same sartorius muscle The action potential traces showed prolonged positive after-depolarisation phases lasting well beyond 20 ms following the surface action potential deflections -LRB- Fig. 1Aa -RRB- , consistent with persistent excitation of an intact transverse -LRB- T - -RRB- tubular system -LRB- Adrian and Peachey 1973 -RRB- . These persisted both in the hyperosmotic solution -LRB- Fig. 1Ab -RRB- and the iso-osmotic solution to which the fibres were finally returned -LRB- Fig. 1Ac -RRB- , confirming a persistence in transverse tubular excitation and its excitation following initiation of the surface component of the action potential through these manipulations . Use of 600 mOsm as opposed to standard Ringer increased the action potential latencies and consequently the calculated values of conduction velocity . Thus , conduction velocities in the initial iso-osmotic Ringer solution , then at 600 mOsm-Ringer solution and finally , the returned iso-osmotic Ringer were 0.98 ± 0.092 m s − 1 -LRB- n = 11 fibres -RRB- , 0.58 ± 0.051 m s − 1 -LRB- n = 10 -RRB- and 0.72 ± 0.101 m s − 1 -LRB- n = 6 -RRB- respectively . The effect of hyperosmolarity on conduction velocity was thus at least partly reversible . However , this decrease in conduction velocity with increased osmolarity -LRB- from 250 to 600 mOsm -RRB- was not accompanied by qualitative changes in action potential or dV/dt waveform . Likewise , the waveforms showed no qualitative changes following return to the iso-osmotic Ringer solution after hyperosmotic exposure . Grading of conduction velocity changes with extracellular osmolarity Figure 2 summarises typical traces of action potentials -LRB- column A -RRB- and dV/dt -LRB- column B -RRB- for experiments that systematically investigated the effects of graded changes in extracellular osmolarity reflecting different extracellular sucrose concentrations on conduction velocity , action potential waveform , and dV/dt obtained at 7 °C . Action potential waveforms again included both early rapid surface action potential deflections following the stimulus artefact and prolonged after-depolarisation phases that lasted well beyond 20 ms , reflecting T-tubular activation , at all the extracellular osmolarities studied -LRB- cf. Adrian and Peachey 1973 -RRB- . Measurements of latencies between the stimulus artefacts to the maximum value of the dV/dt traces then suggested a noticeable overall decrease in the calculated conduction velocities with increasing osmolarity . All these changes occurred in the absence of any excessive depolarization in resting membrane potential over the range of explored osmolarities . Thus resting potential at an osmolarity of 250 mOsm was − 85.27 ± 1.05 mV -LRB- n = 87 -RRB- ; that at an osmolarity of 700 mOsm was − 75.94 ± 2.20 mV -LRB- n = 33 -RRB- . However , resting potential at an osmolarity of 750 mOsm was − 62.71 ± 2.82 mV -LRB- n = 17 -RRB- -LRB- cf. Fraser et al. 2006 -RRB- . At the latter extracellular osmolarity a small proportion of action potentials appeared to display markedly reduced amplitudes resulting in peak deflections that failed to show overshoots , and a firing of two consecutive action potentials after a single stimulation in the case of one fibre . Accordingly , the investigations were not performed at higher sucrose concentrations.Fig . 2Typical action potential -LRB- A -RRB- and dV/dt -LRB- B -RRB- traces recorded in the following extracellular osmolarities : -LRB- a -RRB- 250 , -LRB- b -RRB- 400 , -LRB- c -RRB- 500 , -LRB- d -RRB- 600 , -LRB- e -RRB- 650 and -LRB- f -RRB- 750 -LRB- all values given in mOsm -RRB- . There is a notable overall increase in the latencies measured from -LRB- B -RRB- Plots of conduction velocity and maximum dV/dt with increasing extracellular osmolarity Figure 3 summarizes the results of systematic studies of the dependence of conduction velocity on extracellular osmolarity in a statistically larger number of muscle fibres . Action potentials and their corresponding dV/dt were measured systematically in n = 87 , 18 , 27 , 11 , 10 , 33 and 13 fibres at sucrose concentrations of 0 , 150 , 250 , 350 , 400 , 450 and 500 mM corresponding in turn to osmolarities of 250 , 400 , 500 , 600 , 650 , 700 and 750 mOsm respectively . Figure 3 demonstrates that both conduction velocity -LRB- a -RRB- and maximum dV/dt -LRB- means ± standard errors of the mean -RRB- -LRB- b -RRB- monotonically decreased with extracellular osmolarity . Figure 3a then compares these experimental values of conduction velocity against predictions from the two hypotheses for the dependence of Ri on osmolarity . In both cases , θ was calculated from Ri and the extracellular osmolarity , osm , using the relationship derived from Eqs . -LRB- 4 -RRB- and -LRB- 5 -RRB- above that : Fig. 3Experimentally measured conduction velocities -LRB- a -RRB- and maximum dV/dt -LRB- mean ± standard error of the mean -RRB- plotted against extracellular osmolarity -LRB- ◆ -RRB- . Dashed lines in -LRB- a -RRB- : velocities predicted for a situation in which Ri decreases proportionally as a result of increased intracellular ionic strength produced by changes in cell volume brought about by the osmolarity changes . Continuous line : predictions when Ri increases with increasing extracellular osmolarity suggested by Hodgkin and Nakajima -LRB- 1972 -RRB- . The changes in -LRB- a -RRB- were accompanied by decreases in maximum dV/dt -LRB- b -RRB- with increasing osmolarity , as expected for propagation brought about by local circuit currents The dotted line denotes expectations from the first situation outlined above in which Ri decreases proportionally with increasing extracellular osmolarity : this predicts that conduction velocity does not alter with osmolarity from its control value in iso-osmotic solutions corresponding to a constant value of 0.85 m s − 1 obtained in iso-osmotic solutions . The continuous line represents the second hypothesis in which Ri depends both on temperature and extracellular osmolarity as reported by Hodgkin and Nakajima -LRB- 1972 -RRB- . The function displayed assumes the sarcoplasmic conductivity Gi -LRB- = 1/Ri -RRB- to have a Q10 = 1.37 , and a value of Ri at 2 °C , of 298.51 Ω cm in a muscle fibre within an extracellular solution of 250 mOsm osmolarity and of 390.63 Ω cm in a muscle fibre within an extracellular solution of 600 mOsm osmolarity , as suggested by Hodgkin and Nakajima -LRB- 1970 , 1972 -RRB- . Then , let θ1 , Ri1 and osm1 correspond to conditions in iso-osmotic Ringer and θ2 , Ri2 and osm2 correspond to conditions in any hyperosmotic solution . Thus : Since θ1 = 0.85 ± 0.029 m s − 1 in iso-osmotic solution , where The values generated by the above equations predicted a decline in conduction velocity with increasing extracellular osmolarity . To test the statistical significance of the goodness-of-fit of the two contrasting predictions to the experimental data obtained , an Fx test that takes into account the difference of the two reduced-χ2 values in proportion to the first χ2 term was performed -LRB- Bevington 1969 -RRB- . This derived for each hypothesis a value of χ2 depicting the summed squared deviation of the original data , yi , to the predicted findings , y -LRB- xi -RRB- , obtained at any extracellular osmolarity , xi , such that : Values for χ2 obtained in the two cases , χ12 where the predicted conduction velocity is a constant value and χ22 where there is an increase in Ri with extracellular osmolarity were 21.360 and 15.141 respectively . These values were then used to compute a F-statistic , given by : where n , the sample size = 200 and n − 1 is the number of degrees of freedom . The reduced-χ2 tests for goodness-of-fit yielded a F-statistic of 81.73 consistent with a significantly better fit -LRB- P << 0.001 -RRB- to predictions from a situation in which Ri increased as described by Hodgkin and Nakajima -LRB- 1972 -RRB- rather than a decrease in Ri with increasing extracellular osmolarity . Discussion This paper begins from classic analyses -LRB- Hodgkin 1954 ; Adrian 1975 -RRB- of the dependence of action potential conduction velocities upon the diameter of nerve fibres studied in large volume conductors under iso-osmotic extracellular conditions . This had employed cable analysis that attributed these propagation processes to local circuit currents driven by Na + currents , INa , generating the rising phase of the action potential that in turn discharged initially passive circuit components -LRB- Valdiosera et al. 1974 -RRB- equivalent to a membrane capacitance per unit area , Cm and resistance Rm of unit membrane area in series with an axial cytoplasmic resistance per unit length , ri . The latter is related to fibre radius a and sarcoplasmic resistivity Ri -LRB- kΩ cm -RRB- by Ri = riπa2 giving the original result that conduction velocity would vary as the square root of a . The present paper extends this analysis to the effects of varying extracellular osmolarity on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle , a situation that differed in a number of respects . First , skeletal muscle contains excitable transverse -LRB- T - -RRB- tubular membrane system responsible for initiating excitation-contraction coupling -LRB- Adrian et al. 1969 , 1970 ; Huang 1993 ; Nielsen et al. 2004 ; Stephenson 2006 -RRB- . Nevertheless , the rapid initial sarcolemmal membrane activation that ensures rapid action potential propagation producing a synchronous sarcomeric activation likely largely precedes full tubular excitation -LRB- Adrian and Peachey 1973 -RRB- . Recent detubulation experiments left surface membrane conduction velocities unchanged suggesting a separation of surface and T-tubule excitation , the latter possibly initiated separately by Na + channels localized around the T-tubular luminal mouths -LRB- Sheikh et al. 2001 -RRB- . Increasing extracellular osmolarity did not increase the diameter of the necks of the T-tubules important in electrical connectivity between T-tubule network and the fibre membrane -LRB- Launikonis and Stephenson 2002 , 2004 -RRB- . These findings would permit muscle fibre conduction velocities to be analysed in terms of surface cylinders . Second , skeletal muscle volume alters inversely with extracellular osmolarity -LRB- Blinks 1965 ; Ferenczi et al. 2004 -RRB- in turn correspondingly increasing solute concentration , in contrast to the situation represented by comparisons of nerve fibres of different diameters in similarly iso-osmotic extracellular solutions . This paper then derived quantitative consequences from two possible hypotheses emerging from the above conditions . On the one hand , classical electrolyte theory -LRB- Robinson and Stokes 1959 ; Atkins 1998 -RRB- predicts a specific conductance Ksp attributable to each intracellular ion species increasing proportionally with solute concentration following decreases in cell volume in hyperosmotic solution . This led to a prediction of a decrease in Ri precisely correcting out effects of any osmotically induced diameter change together leaving conduction velocity constant . On the other hand , empirical observations suggesting increases in Ri with extracellular osmolarity -LRB- Hodgkin and Nakajima 1972 -RRB- permitted construction of a quantitative formula for the resulting variations in Ri with extracellular osmolarity as well as temperature . This led to predictions of a conduction velocity that decreased with increasing extracellular osmolarity . The experiments described in this paper then sought to distinguish between the two hypotheses . It investigated the effects of extracellular osmolarity on conduction velocities of surface membrane action potentials in surface fibres , that would be maximally exposed to these solution changes , in frog skeletal muscle , following stimulation at a Vaseline seal at defined distances from the microelectrode recording site . Simultaneous records were made of the rate of change of membrane potential dV/dt that would be provided a consistent time point at which there would be a maximal action potential slope as well as rate of discharge of the membrane capacitance , Cm , by local currents driven by the Na + current INa . These studies showed that conduction velocity declined with increasing extracellular osmolarity along with maximum dV/dt as expected for a process dependent upon a local circuit current flow , despite relatively constant resting membrane potentials . These changes in conduction velocity in hyperosmotic solution were at least partially reversible . Furthermore , there were no changes in the nature of the action potential waveforms including early surface deflections and late after-depolarisation phases observed , consistent with a minimal change in the capacity for T-tubular excitation , through a still intact tubular system , that nevertheless followed generation of the initial surface component of the action potential wave . Further systematic study in larger fibre numbers through a range of osmolarities -LRB- 250 -- 750 mOsm -RRB- demonstrated corresponding declines in conduction velocity and maximum dV/dt . At the highest osmolarity -LRB- 750 mOsm -RRB- , a small proportion of fibres variously showed low action potential overshoots which however did not correlate with the situations where there was a reduced conduction velocity , as well as multiple firing in a single muscle fibre . Nevertheless , further studies were not made at these and higher osmolarities ; in any case , muscle fibres are thought only to act close to perfect osmometers at osmolarities up to around four times that of the iso-osmotic solution -LRB- Blinks 1965 -RRB- . These findings are thus consistent with the second hypothesis in which sarcoplasmic resistivity , Ri , increases with the fibre shrinkage observed in hyperosmotic solutions , as suggested by Hodgkin and Nakajima -LRB- 1972 -RRB- , as opposed to the first possibility outlined above in which conduction velocity should be constant . This was borne out by objective statistical analysis of the goodness-of-fit of the predictions derived from the two contrasting hypotheses as expressed in their resulting reduced-χ2 values , with the experimental values of conduction velocity generally assuming slightly higher values than predicted . The latter might reflect minor departures from a purely continuous conduction as postulated for unmyelinated cylindrical fibres , either due to contributions from peripheral regions of tubular membrane less isolated than the remaining tubular system -LRB- Hodgkin and Nakajima 1972 -RRB- , or the clustering of sodium channels around the tubular necks as reported by Sheikh et al. -LRB- 2001 -RRB- that may contribute to a more saltatory-like conduction in the muscle that would be expected to generally speed up propagation of electrical activity . Electronic supplementary material Below is the link to the electronic supplementary material . 49 kb doc

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

conduction velocity
osmolarity
skeletal muscle