Silver

diagnosis

in

neuropathology

principles

practice

and

revised

interpretation

Silver-staining

methods

are

helpful

for

histological

identification

of

pathological

deposits

In

spite

of

some

ambiguities

regarding

their

mechanism

and

interpretation

they

are

widely

used

for

histopathological

diagnosis

In

this

review

four

major

silver-staining

methods

modified

Bielschowsky

Bodian

Gallyas

-LRB-

GAL

-RRB-

and

Campbell

Switzer

-LRB-

CS

-RRB-

methods

are

outlined

with

respect

to

their

principles

basic

protocols

and

interpretations

thereby

providing

neuropathologists

technicians

and

neuroscientists

with

a

common

basis

for

comparing

findings

and

identifying

the

issues

that

still

need

to

be

clarified

Some

consider

argyrophilia

to

be

a

homogeneous

phenomenon

irrespective

of

the

lesion

and

the

method

Thus

they

seek

to

explain

the

differences

among

the

methods

by

pointing

to

their

different

sensitivities

in

detecting

lesions

-LRB-

quantitative

difference

-RRB-

Comparative

studies

however

have

demonstrated

that

argyrophilia

is

heterogeneous

and

dependent

not

only

on

the

method

but

also

on

the

lesion

-LRB-

qualitative

difference

-RRB-

Each

staining

method

has

its

own

lesion-dependent

specificity

and

within

this

specificity

its

own

sensitivity

This

method

and

lesion-dependent

nature

of

argyrophilia

enables

operational

sorting

of

disease-specific

lesions

based

on

their

silver-staining

profiles

which

may

potentially

represent

some

disease-specific

aspects

Furthermore

comparisons

between

immunohistochemical

and

biochemical

data

have

revealed

an

empirical

correlation

between

GAL

CS-deposits

and

-LRB-

-RRB-

tau

-LRB-

corticobasal

degeneration

progressive

supranuclear

palsy

and

argyrophilic

grains

-RRB-

and

its

complementary

reversal

between

GAL

CS

deposits

and

-LRB-

-RRB-

tau

-LRB-

Pick

bodies

-RRB-

Deposits

containing

both

and

tau

-LRB-

neurofibrillary

tangles

of

Alzheimer

type

-RRB-

are

GAL

CS

Although

no

molecular

explanations

other

than

these

empiric

correlations

are

currently

available

these

distinctive

features

especially

when

combined

with

immunohistochemistry

are

useful

because

silver-staining

methods

and

immunoreactions

are

complementary

to

each

other

Introduction

The

year

was

the

centenary

anniversary

of

the

first

clinical

description

of

a

presenile

patient

with

progressive

cognitive

decline

later

defined

as

Alzheimer

's

disease

-LRB-

AD

-RRB-

-LSB-

-RSB-

It

was

the

same

year

-LRB-

-RRB-

when

Santiago

Ramón

y

Cajal

and

Camillo

Golgi

were

awarded

the

Nobel

prize

for

their

contributions

on

neuroanatomy

achieved

by

using

silver-staining

methods

especially

Golgi-silver

technique

In

the

field

of

neuropathology

as

well

silver

staining

was

the

principal

method

of

choice

at

that

time

which

visualized

senile

plaques

-LRB-

SPs

-RRB-

neurofibrillary

tangles

-LRB-

NFTs

-RRB-

and

Pick

bodies

-LSB-

-RSB-

More

than

years

have

passed

before

biochemical

compositions

of

these

pathological

structures

were

unveiled

During

this

period

technical

trials

to

identify

and

localize

these

molecules

on

histological

sections

showed

the

effectiveness

of

immunohistochemistry

and

revolutionized

not

only

research

but

also

routine

diagnosis

in

the

field

of

neuropathology

Probably

because

of

the

apparent

success

in

the

molecular

explanation

of

diseases

recent

attention

to

silver

staining

has

not

been

very

enthusiastic

Although

most

silver-staining

methods

are

still

left

with

some

ambiguities

neuropathologists

have

profited

by

their

efficacies

Silver-staining

methods

have

been

steadily

improved

and

one

of

the

recent

advances

is

undoubtedly

Gallyas

method

-LSB-

-RSB-

In

practice

it

allows

stable

reproducible

and

sensitive

visualization

with

low

background

However

it

is

unfortunate

that

their

principles

technical

details

and

interpretations

which

are

different

according

to

each

method

are

sometimes

not

sufficiently

shared

among

neuropathologists

and

technicians

It

frequently

happens

that

some

time-honored

protocols

are

left

without

possible

explanations

and

their

interpretation

is

often

restricted

to

argyrophilic

or

not

This

naive

interpretation

is

based

on

the

assumption

that

argyrophilia

is

a

homogeneous

phenomenon

irrespective

of

the

staining

method

and

the

lesion

Recent

studies

have

demonstrated

on

the

contrary

that

argyrophila

is

heterogeneous

and

dependent

on

the

staining

method

and

the

lesion

-LSB-

-RSB-

Here

established

silver-staining

methods

in

the

field

of

neuropathology

are

reconsidered

with

the

intension

of

improving

their

use

and

interpretation

The

first

part

of

this

review

summarizes

basic

principles

and

outlines

protocols

about

four

principal

silver-staining

methods

for

formalin-fixed

paraffin-embedded

samples

from

human

autopsy

brains

mainly

with

degenerative

diseases

currently

in

use

in

our

laboratory

and

their

modifications

Although

other

silver-staining

methods

have

also

been

invented

to

visualize

normal

neuronal

structures

astrocytes

microglia

and

experimental

tissue

samples

it

is

not

possible

to

encompass

the

whole

range

of

these

techniques

mainly

because

of

the

limitation

of

my

own

experience

The

second

part

provides

an

overview

of

similarities

and

differences

between

silver-staining

methods

and

immunohistochemistry

so

that

a

comprehensive

interpretation

including

their

pitfalls

results

Nevertheless

this

review

is

not

intended

to

establish

a

relative

grading

of

these

methods

by

identifying

the

possible

superiority

of

a

method

-LRB-

for

example

immunohistochemistry

-RRB-

over

another

-LRB-

for

example

a

silver-staining

method

-RRB-

Instead

it

is

designed

to

show

how

unique

each

staining

method

can

be

by

providing

a

distinct

point

of

view

each

independent

of

the

other

If

we

are

prepared

to

take

these

possible

differences

of

staining

methods

into

account

such

an

approach

will

provide

a

less

biased

multi-faceted

and

fuller

stereoscopic

picture

than

can

be

achieved

with

immunohistochemistry

alone

General

principle

of

silver

staining

The

coloration

in

conventional

histology

is

based

on

the

selective

affinity

of

a

dye

to

some

tissue

elements

relative

to

surrounding

elements

and

their

permeability

This

differential

affinity

to

specific

tissue

elements

is

finally

represented

by

the

distinctive

original

color

of

the

dye

as

seen

with

its

solution

This

is

essentially

a

single

step

procedure

called

dyeing

and

does

not

involve

chemical

transformation

of

the

dye

Subsequent

washing

step

is

also

selective

in

dissolving

out

the

attached

dye

which

is

called

differentiation

-LSB-

-RSB-

Various

combinations

-LRB-

for

example

hematoxylin

and

eosin

the

Klüver-Barrera

method

etc.

-RRB-

are

currently

in

use

for

neuropathological

examinations

The

mechanism

of

silver

staining

is

however

completely

different

Because

the

solutions

containing

coloring

agents

used

for

silver-staining

methods

are

usually

colorless

simple

application

of

these

solutions

to

tissue

sections

by

itself

does

not

allow

visualization

through

light

microscope

even

if

the

reagent

has

some

affinity

to

the

tissue

elements

Subsequent

chemical

transformation

is

necessary

for

visualization

In

silver

solutions

most

of

the

silver

is

present

as

ion

or

complex

salt

The

silver

ions

should

be

first

attached

to

the

target

elements

as

silver

ions

or

silver

salts

-LRB-

step

-RRB-

This

attachment

however

does

not

allow

visualization

by

itself

The

attached

silver

ions

-LRB-

Ag

-RRB-

have

to

be

reduced

-LRB-

accept

electrons

-RRB-

to

yield

in

situ

metallic

silver

particles

-LRB-

Ag

-RRB-

now

visualized

only

if

their

amount

and

size

are

sufficient

for

microscopic

detection

-LRB-

step

-RRB-

Therefore

not

only

the

differential

affinity

of

the

silver

ion

to

some

tissue

elements

-LRB-

step

-RRB-

but

also

its

chemical

transformation

for

visualization

-LRB-

step

-RRB-

is

essential

Although

the

attachment

of

silver

ions

to

tissue

elements

is

based

on

the

in

situ

formation

of

silver

salt

these

silver

ions

either

in

the

solution

or

as

salts

are

reducible

They

undergo

reduction

to

form

metallic

silver

Gallyas

demonstrated

that

there

is

a

time

lag

between

the

rapid

attachment

of

silver

ions

onto

the

section

-LRB-

step

within

min

-RRB-

and

delayed

formation

of

metallic

silver

particles

-LRB-

step

-RRB-

there

-LSB-

-RSB-

Because

these

two

steps

are

independently

affected

by

pH

temperature

the

concentration

of

silver

ion

and

coexisting

molecules

-LSB-

-RSB-

these

steps

are

independent

even

if

both

take

place

in

a

single

manipulation

This

dual

function

of

silver

that

enables

site-directed

chemical

transformation

is

fundamental

to

various

silver-staining

methods

Because

some

tissue

elements

-LRB-

glutathione

creatine

adrenaline

etc.

-RRB-

exhibit

an

intrinsic

reducing

capacity

which

may

be

strong

enough

to

reduce

silver

ions

into

metallic

silver

particles

in

situ

these

two

steps

-LRB-

selective

attachment

and

visualization

after

reduction

of

silver

ions

-RRB-

may

take

place

in

a

single

manipulation

This

is

called

silver

reaction

or

argentaffin

reaction

-LSB-

-RSB-

When

the

reducing

capacity

of

the

tissue

elements

is

absent

or

not

sufficient

it

is

necessary

to

provide

reducing

agents

externally

after

silver

ions

have

been

attached

to

the

tissue

elements

It

is

also

possible

to

facilitate

or

modify

these

steps

by

some

reagents

-LSB-

-RSB-

Anyway

this

two-step

procedure

is

called

postblackening

-LRB-

Nachschwärzen

-RRB-

or

argentophilic

reaction

-LSB-

-RSB-

and

most

of

the

current

silver-staining

methods

for

neuropathology

consist

in

essence

of

at

least

these

two

steps

If

sufficient

amount

of

silver

ions

have

been

attached

to

the

tissue

elements

but

not

have

been

reduced

spontaneously

after

step

it

is

possible

to

visualize

them

simply

by

applying

an

appropriate

reducing

agent

externally

This

is

called

chemical

development

during

which

the

amount

of

deposited

silver

in

the

tissue

section

remains

unchanged

However

it

is

not

easy

to

increase

the

amount

of

silver

deposits

during

step

For

example

increasing

the

concentration

of

silver

reagents

or

prolongation

of

incubation

time

is

frequently

hampered

by

nonspecific

staining

This

trade

off

between

sensitivity

and

specificity

principally

encountered

in

all

kinds

of

staining

is

especially

troublesome

with

silver-staining

methods

because

reactions

in

the

silver

solutions

and

those

in

reducing

agents

are

both

capricious

and

difficult

to

standardize

This

is

one

of

the

reasons

why

more

sensitive

stable

and

reproducible

silver

solutions

and

staining

methods

have

been

a

matter

of

considerable

endeavor

for

a

long

period

as

will

be

discussed

later

Particle

size

and

possible

determinants

of

selective

staining

It

has

been

reported

that

the

apparent

color

of

silver-stained

elements

is

correlated

with

the

size

of

final

silver

particles

In

the

test

tube

the

smallest

range

in

their

diameter

-LRB-

nm

-RRB-

is

correlated

with

yellow

color

The

largest

range

-LRB-

nm

-RRB-

is

associated

with

black

color

Intermediate

ranges

are

correlated

with

red

-LRB-

nm

-RRB-

or

blue-purple

-LRB-

nm

-RRB-

-LSB-

-RSB-

as

reported

by

Liesegang

-LSB-

-RSB-

The

emission

spectra

of

fluorogenic

semiconductor

nanocrystals

recently

introduced

for

fluorolabeling

are

dependent

not

only

on

their

materials

but

also

on

their

particle

size

-LSB-

-RSB-

Although

the

size

range

of

semiconductor

nanocrystals

-LRB-

nm

-RRB-

is

much

smaller

than

that

of

silver

particles

it

is

interesting

to

note

that

these

differences

in

their

color

spectra

are

dependent

on

the

particle

size

in

both

silver

particles

and

the

nanocrystals

Others

reported

however

that

black

color

is

correlated

with

a

larger

amount

of

deposits

-LSB-

-RSB-

Although

the

deposition

of

silver

particles

is

primed

by

an

affinity

of

the

silver

ion

to

some

tissue

elements

is

this

affinity

the

only

major

determinant

of

the

selective

staining

According

to

Seki

-LSB-

-RSB-

some

colloidal

substance

in

the

tissue

may

hinder

formation

and

deposition

of

metallic

silver

particles

-LRB-

Schutzkolloid

-RRB-

In

addition

packing

density

of

tissue

ultrastructures

-LRB-

for

example

fiber

density

-RRB-

may

also

be

responsible

because

an

excessive

looseness

of

the

structure

may

not

allow

secure

settlement

of

silver

particles

It

is

also

possible

that

its

extreme

tightness

does

not

permit

appropriate

growth

of

silver

particles

He

then

concluded

that

a

small

quantity

of

this

intervening

colloid

-LRB-

eine

Armut

an

Schutzkolloid

-RRB-

and

an

appropriate

density

of

the

structure

-LRB-

eine

passende

Strukturdichte

-RRB-

of

the

target

structure

are

also

important

for

the

selectivity

of

silver

staining

This

idea

that

some

spatial

constraints

around

silver

particles

in

addition

to

the

primary

affinity

of

the

silver

ion

are

essential

in

determining

the

selectivity

of

silver

staining

may

be

related

to

the

current

concept

of

disease-specific

conformational

status

of

pathological

deposits

and

their

constituent

molecules

These

explanations

are

however

far

from

complete

for

the

majority

of

silver-staining

methods

Furthermore

because

these

experiments

have

been

performed

mainly

in

animal

tissues

in

which

sampling

and

handling

of

tissue

materials

are

optimal

it

remains

to

be

clarified

whether

similar

explanations

are

applicable

to

human

samples

obtained

for

neuropathological

examinations

often

containing

distinct

pathological

lesions

and

suffering

from

suboptimal

handling

such

as

postmortem

delay

suboptimal

fixation

and

embedding

in

paraffin

wax

Even

though

various

methods

for

celloidin

or

paraffin-embedded

autopsy

samples

have

been

invented

Table

shows

fundamental

steps

and

contents

of

the

solutions

for

the

silver-staining

methods

currently

in

use

in

our

laboratory

Table

for

different

silver-staining

methodsBielschowsky

method

-LRB-

original

-RRB-

for

frozen

sections

-LSB-

-RSB-

Modified

Bielschowsky

method

for

paraffin

sections

-LSB-

-RSB-

Bodian

method

-LSB-

-RSB-

Gallyas

method

-LSB-

-RSB-

Campbell

Switzer

method

-LSB-

-RSB-

PretreatmentPyridine

-LRB-

h

-RRB-

potassium

permanganate

-LRB-

RT

min

-RRB-

oxalic

acid

-LRB-

RT

min

-RRB-

-LSB-

-RSB-

lanthanum

sodium

acetate

-LRB-

RT

min

-RRB-

-LSB-

-RSB-

WashingDW

DW

Silver

solution

silver

nitrate

-LRB-

RT

h

-RRB-

silver

nitrate

-LRB-

RT

min

-RRB-

silver

proteinate

metallic

copperf

-LRB-

°C

h

-RRB-

Alkaline

silver

iodate

-LRB-

RT

min

-RRB-

silver

nitrate

potassium

iodide

sodium

hydroxidePyridine

silver

-LRB-

RT

min

-RRB-

silver

nitrate

-LRB-

volume

-RRB-

pyridine

potassium

acetic

acid

min

acetic

acid

min

timesSilver

solution

b

ammoniacal

c

ammoniacal

silver

-LRB-

RT

min

in

the

dark

-RRB-

Physical

developerg

-LRB-

RT

min

-RRB-

silver

nitrate

tungsto-silicic

acid

ammonium

nitrate

sodium

carbonate

formalinPhysical

developergWashingDWDW

ammoniad

Chemical

formalin

in

tap

waterDevelopere

in

ammoniacal

silver

sodium

sulfiteDW

distilled

water

RT

room

formalin

in

tap

water

is

optional

-LSB-

-RSB-

ml

of

silver

nitrate

five

drops

of

sodium

hydroxide

followed

by

titration

with

ammonia

to

dissolve

the

precipitates

DW

-LRB-

ml

-RRB-

is

then

added

-LSB-

-RSB-

cAmmonium

hydroxide

was

added

to

silver

nitrate

drop

by

drop

until

the

precipitates

turn

clear

Additional

two

drops

of

ammonium

hydroxide

is

recommended

-LSB-

-RSB-

dAdd

three

drops

of

ammonium

hydroxide

to

DW

-LSB-

-RSB-

eAdd

three

drops

of

the

developer

-LRB-

formalin

ml

DW

ml

concentrated

nitric

acid

one

drop

citric

acid

g

-RRB-

to

the

ammoniacal

silver

used

as

silver

solution

II

-LSB-

-RSB-

g

of

metallic

copper

in

ml

of

the

silver

proteinate

solution

is

optimal

-LSB-

-RSB-

gThe

physical

developers

reported

for

Gallyas

and

Campbell

Switzer

methods

are

essentially

identical

In

our

laboratory

equal

amount

of

stock

solutions

A

-LRB-

g

sodium

carbonate-anhydrous

ml

DW

-RRB-

and

B

-LRB-

g

silver

nitrate

g

tungusto-silicic

acid

g

ammonium

nitrate

formalin

ml

ml

DW

-RRB-

are

mixed

immediately

before

use

Bielschowsky

methods

Improvements

in

silver-staining

methods

have

often

been

derived

from

chemistry

The

ammoniacal

silver

solution

had

been

used

for

production

of

mirror

based

on

the

nature

of

the

complex

silver

diamine

-LSB-

Ag

-LRB-

-RRB-

-RSB-

in

the

solution

that

is

liable

to

precipitate

after

being

reduced

at

the

interface

between

the

glass

surface

and

the

solution

This

is

called

the

silver

mirror

reaction

and

it

was

Fajersztajn

who

initially

applied

this

phenomenon

to

tissue

staining

-LSB-

-RSB-

Later

Max

Bielschowsky

-LSB-

-RSB-

improved

this

method

by

incubating

formalin-fixed

frozen

sections

in

the

silver

nitrate

prior

to

the

ammoniacal

silver

solution

-LRB-

Table

-RRB-

Although

the

exact

mechanism

still

remains

to

be

clarified

-LSB-

-RSB-

it

is

plausible

that

this

prior

treatment

with

the

silver

nitrate

provides

active

foci

for

subsequent

silver

deposition

in

the

ammoniacal

silver

solution

-LSB-

-RSB-

Anyway

it

is

reasonable

to

suppose

that

this

stepwise

impregnation

-LRB-

silver

nitrate-ammoniacal

silver

-RRB-

improved

the

staining

probably

by

increasing

the

amount

and

the

size

of

silver

precipitates

-LSB-

-RSB-

Early

modifications

of

Bielschowsky

method

were

summarized

by

Beech

and

Davenport

-LSB-

-RSB-

Garven

and

Gairns

-LSB-

-RSB-

provided

a

comprehensive

review

on

the

possible

mechanism

of

the

so-called

Bielschowsky-Gros

method

and

its

modifications

The

presence

of

a

huge

number

of

modifications

however

indicates

that

this

method

is

highly

unstable

and

still

awaits

improvements

A

modification

for

paraffin-embedded

samples

of

normal

neural

tissues

from

various

animal

species

was

first

proposed

by

Sevier

and

Munger

-LSB-

-RSB-

Practically

it

is

probably

Yamamoto

Hirano

's

modification

-LSB-

-RSB-

which

is

currently

accepted

as

the

standard

Bielschowsky

method

for

neuropathological

examination

of

paraffin-embedded

autopsy

samples

-LRB-

Table

-RRB-

This

Yamamoto

Hirano

's

modification

is

different

from

other

conventional

modifications

of

Bielschowsky

method

because

the

usual

silver

nitrate

ammoniacal

silver

sequence

is

followed

by

another

ammoniacal

silver

solution

containing

developers

Bodian

method

Bodian

established

a

new

method

applicable

to

paraffin-embedded

sections

for

staining

nerve

fibers

and

nerve

endings

-LSB-

-RSB-

This

method

is

rather

stable

and

reproducible

and

is

currently

in

use

for

neuropathology

He

used

silver

proteinate

-LRB-

which

usually

contains

of

silver

-RRB-

solution

and

placed

metallic

copper

in

it

Because

the

staining

does

not

work

well

in

the

absence

of

metallic

copper

he

speculated

that

metallic

copper

might

regulate

the

deposition

of

metallic

silver

by

decreasing

the

concentration

of

silver

ion

-LSB-

-RSB-

However

the

mixture

of

silver

nitrate

and

powdered

egg

albumin

was

reportedly

successful

even

in

the

absence

of

metallic

copper

-LSB-

-RSB-

Nevertheless

the

measured

concentration

of

silver

ion

gradually

decreases

after

addition

of

metallic

copper

as

demonstrated

by

two

independent

groups

-LSB-

-RSB-

Concomitant

and

gradual

increase

in

the

concentration

of

copper

ion

-LSB-

-RSB-

is

accompanied

by

a

decrease

in

pH

-LSB-

-RSB-

These

reciprocal

changes

between

concentrations

of

silver

and

copper

ions

are

mainly

explained

by

a

primary

difference

in

ionization

energy

between

copper

and

silver

In

other

words

copper

is

more

liable

to

be

ionized

than

silver

Because

their

concentrations

are

not

influenced

by

the

presence

of

sections

in

the

solution

the

amount

of

silver

ion

trapped

in

the

section

is

practically

negligible

While

the

addition

of

metallic

copper

up

to

ml

progressively

decreases

the

concentration

of

silver

ion

in

dose-dependent

fashion

-LRB-

from

to

μg/ml

-RRB-

further

increase

in

the

amount

of

metallic

copper

up

to

ml

does

not

lead

to

further

increase

in

the

copper

ion

nor

further

decrease

in

the

silver

ion

in

the

solution

-LSB-

-RSB-

Indeed

this

amount

of

copper

ml

is

in

perfect

agreement

of

the

amount

ml

originally

recommended

by

Bodian

-LSB-

-RSB-

and

the

amount

of

metallic

copper

more

or

less

than

this

range

does

not

give

optimal

results

Interestingly

not

only

the

amount

of

metallic

copper

but

also

the

shape

of

metallic

copper

-LRB-

foil

plate

chip

or

granules

-RRB-

influences

the

concentration

of

copper

and

silver

ions

and

the

results

of

staining

The

metallic

copper

as

foil

is

correlated

with

the

highest

concentration

of

copper

ion

and

the

most

intense

staining

-LSB-

-RSB-

Moreover

these

reactions

in

the

silver

proteinate

solution

occur

slowly

and

the

equilibrium

is

reached

after

h.

Figure

illustrates

putative

chronological

events

during

incubation

in

the

silver

proteinate

solution

and

development

In

the

early

phase

of

silver

impregnation

the

high

concentration

of

silver

ion

facilitates

its

attachment

to

the

section

-LRB-

step

Fig.

-RRB-

This

initial

attachment

is

apparently

not

selective

because

early

interruption

of

incubation

yields

an

overall

nonselective

nerve

staining

-LSB-

-RSB-

Gradual

deposition

of

silver

ion

as

metallic

silver

on

the

surface

of

metallic

copper

-LRB-

step

Fig.

-RRB-

leads

to

a

gradual

decrease

in

the

concentration

of

silver

ion

and

a

concomitant

increase

in

the

concentration

of

copper

ion

in

the

solution

Continued

incubation

in

the

solution

with

a

decreasing

concentration

of

silver

ion

facilitates

liberation

of

the

silver

ion

once

trapped

in

the

section

back

into

the

solution

-LRB-

step

Fig.

-RRB-

The

selective

staining

of

Bodian

method

is

explained

if

this

rate

of

liberation

of

silver

ion

back

to

the

solution

-LRB-

step

Fig.

-RRB-

is

different

according

to

the

tissue

elements

This

chronological

change

may

be

necessary

to

yield

the

selective

staining

and

explains

why

an

equivalent

concentration

of

copper

ion

provided

as

copper

sulfate

instead

of

metallic

copper

does

not

yield

similar

results

-LSB-

-RSB-

Some

copper

ions

are

also

deposited

onto

the

section

-LRB-

step

Fig.

-RRB-

Residual

silver

proteinate

as

colloidal

fraction

may

release

silver

ions

gradually

throughout

the

incubation

-LRB-

step

Fig.

-RRB-

For

practice

-LRB-

-RRB-

g

of

metallic

copper

as

foil

in

ml

of

silver

proteinate

solution

and

-LRB-

-RRB-

incubation

for

h

are

recommended

as

optimal

for

Bodian

method

-LSB-

-RSB-

Fig.

of

the

Bodian

method

Step

Attachment

of

silver

ions

to

the

section

driven

by

high

concentration

of

silver

ions

single

asterisk

a

fraction

of

silver

ions

are

already

reduced

to

metallic

silver

even

before

chemical

development

Step

Deposition

of

silver

ions

as

metallic

silver

leads

to

a

decrease

in

the

concentration

of

silver

ion

and

reciprocal

liberation

of

copper

ions

Step

Delayed

liberation

of

silver

ions

from

the

section

back

to

the

solution

with

decreased

concentration

of

silver

ion

after

step

Step

Copper

ions

are

now

available

in

the

solution

after

step

to

be

attached

on

the

section

Step

Silver

proteinate

gradually

releases

silver

ions

during

steps

Step

Not

only

silver

but

also

copper

ions/salts

on

the

section

are

reduced

to

metallic

particles

Step

These

metallic

particles

are

replaced

with

metallic

gold

double

asterisk

retrieval

of

copper

ions

in

the

gold

chloride

solution

indicates

that

metallic

copper

has

been

deposited

on

the

section

-LRB-

steps

and

-RRB-

Symbols

circle

silver

square

copper

pentagon

gold

blank

symbols

reducible

-LRB-

ion/salts

-RRB-

filled

symbols

metallic

After

being

incubated

in

the

silver

proteinate

solution

sections

are

treated

with

hydroquinone

with

sodium

sulfite

for

reduction

-LRB-

step

Fig.

-RRB-

-LSB-

-RSB-

It

has

been

reported

that

formaldehyde

suitable

for

reducing

the

silver

diamine

of

Bielschowsky

method

is

not

appropriate

by

itself

for

reducing

the

silver

ions

in

Bodian

method

In

contrast

hydroquinone

suitable

to

reduce

the

silver

ions

in

Bodian

method

is

not

appropriate

for

reducing

the

silver

diamine

of

Bielschowsky

method

-LSB-

-RSB-

However

some

recommended

formaldehyde

in

combination

with

hydroquinone

for

the

reducing

step

of

Bodian

method

-LSB-

-RSB-

Because

it

is

usually

after

the

incubation

in

the

developer

when

the

sections

appear

contrasted

for

visualization

it

seems

reasonable

to

suppose

that

deposited

silver

ions

are

reduced

to

form

metallic

silver

only

after

exposure

to

hydroquinone

If

this

is

the

case

treatment

of

the

silver

impregnated

sections

with

sodium

sulfite

which

dissolves

out

silver

salt

but

not

metallic

silver

will

diminish

the

staining

On

the

contrary

Peters

-LSB-

-RSB-

demonstrated

the

presence

of

sodium

sulfite-resistant

metallic

silver

and

suggested

that

at

least

some

fraction

of

silver

ions

have

already

been

reduced

to

metallic

silver

during

incubation

in

the

silver

proteinate

solution

-LRB-

asterisk

step

Fig.

-RRB-

They

may

serve

as

nuclei

that

attract

surrounding

reducible

silver

ions/salts

to

form

larger

metallic

silver

particles

during

subsequent

development

Indeed

Gallyas

demonstrated

that

radiolabeled

silver

ions

in

the

silver

proteinate

solution

are

deposited

to

the

section

in

the

form

of

metallic

silver

even

before

the

development

This

is

based

on

the

finding

that

the

radiolabeled

silver

on

the

section

is

resistant

to

the

treatment

with

silver

acetic

acid

that

washes

out

reducible

silver

ions

but

not

metallic

silver

particles

Because

the

localization

of

this

radiolabeled

silver

now

deposited

as

metallic

silver

is

in

good

agreement

with

the

final

image

of

the

adjacent

section

stained

by

Bodian

method

it

is

likely

that

these

metallic

silver

deposits

play

a

determinant

role

in

defining

final

image

obtained

with

Bodian

method

-LSB-

-RSB-

At

any

rate

it

is

possible

to

intensify

the

staining

through

subsequent

gold

toning

by

incubating

in

gold

chloride

with

glacial

acetic

acid

-LRB-

three

drops

for

ml

-RRB-

Although

the

mechanism

of

Bodian

method

especially

that

of

copper

ion

is

not

yet

completely

clarified

not

only

silver

but

also

copper

ions

are

retrieved

in

the

gold

chloride

solution

for

toning

-LRB-

double

asterisk

step

Fig.

-RRB-

-LSB-

-RSB-

This

indicates

that

the

ionized

copper

in

the

silver

proteinate

solution

has

been

deposited

on

the

tissue

and

that

the

deposited

copper

in

addition

to

silver

is

replaced

with

gold

-LSB-

-RSB-

This

dual

replacement

may

explain

why

this

gold

toning

is

necessary

and

particularly

effective

for

Bodian

method

After

gold

toning

it

is

now

possible

and

necessary

to

treat

the

sections

with

sodium

thiosulfate

to

dissolve

out

silver

ions

that

remain

unreduced

in

the

tissue

Otherwise

these

remaining

ions

may

be

reduced

after

exposure

to

light

to

form

undesirable

precipitates

Because

Bodian

method

was

originally

invented

to

stain

normal

axons

and

neurofibrils

these

normal

structures

are

always

superimposed

on

pathological

structures

such

as

SPs

and

NFTs

These

intervening

normal

structures

are

one

of

the

major

hindrances

of

Bodian

method

especially

when

examining

silver-stainable

deposits

overlaid

on

normal

structures

Gallyas

method

Another

way

of

improvement

had

been

achieved

based

on

the

chemical

principle

of

photography

physical

development

As

opposed

to

the

chemical

development

additional

silver

ions

are

provided

with

reducing

agents

to

form

silver

particles

around

the

reactive

foci

which

have

been

already

activated

after

exposure

to

light

in

dose-dependent

manner

This

greatly

increases

the

amount

of

metallic

silver

or

oxidized

silver

that

are

now

visible

as

photographic

images

It

was

Liesegang

who

first

introduced

this

idea

to

histological

staining

in

an

attempt

to

establish

a

more

consistent

and

reproducible

method

of

silver

staining

-LSB-

-RSB-

The

protocols

with

physical

development

were

later

customized

successfully

for

neuropathology

by

Gallyas

-LSB-

-RSB-

Because

physical

developer

solutions

contain

in

general

silver

ions

and

reducing

agents

that

together

react

rapidly

protecting

colloid

-LRB-

tungsto-silicic

acid

for

Gallyas

method

-RRB-

is

included

in

order

to

control

by

retarding

this

reaction

-LSB-

-RSB-

As

with

other

silver-staining

methods

the

attachment

of

silver

ion

to

specific

targets

determines

final

localization

of

silver

particles

According

to

Gallyas

this

specific

attachment

is

mediated

by

some

determinant

substance

which

binds

to

the

target

tissue

elements

or

the

tissue

elements

themselves

followed

by

their

chemical

modifications

These

foci

are

enabled

to

attract

silver

ions

to

form

silver

salts

Otherwise

the

determinant

substance

renders

these

foci

capable

of

reducing

silver

ions

to

form

submicroscopic

metallic

silver

particles

bound

to

the

target

elements

The

chemical

nature

of

the

original

determinant

substance

may

determine

which

components

-LRB-

for

example

fibrous

astrocytes

microglia

or

oligodendrocytes

-RRB-

in

the

section

are

finally

silver-stained

-LSB-

-RSB-

For

visualization

of

NFTs

sections

are

incubated

with

silver

iodide

in

alkaline

condition

that

exhibits

affinity

to

NFTs

-LSB-

-RSB-

-LRB-

Table

-RRB-

Although

Gallyas

customized

protocols

according

to

various

target

structures

other

than

NFTs

-LSB-

-RSB-

this

silver

iodide

protocol

for

NFTs

currently

in

routine

use

for

neuropathological

diagnosis

is

defined

in

this

review

as

Gallyas

method

for

simplicity

and

convenience

If

this

silver

solution

is

prepared

with

radiolabeled

silver

it

is

possible

to

quantify

the

silver

ions/particles

after

each

step

of

the

staining

protocol

Gallyas

demonstrated

that

most

of

the

silver

attached

to

the

section

after

incubation

in

this

silver

iodide

solution

is

reducible

silver

salt

but

not

metallic

silver

because

it

is

washed

out

after

incubation

in

nonlabeled

silver

acetic

acid

-LRB-

Table

of

Ref

-LSB-

-RSB-

-RRB-

a

treatment

that

expels

reducible

silver

ions/salts

from

the

sections

Incubation

in

sodium

thiosulfate

or

sodium

cyanide

alternate

ways

to

expel

these

reducible

silvers

diminishes

the

final

image

again

compatible

with

the

assumption

that

the

attached

silver

after

incubation

in

the

silver

iodide

solution

is

silver

ions/salts

but

not

metallic

silver

-LSB-

-RSB-

Is

a

separate

step

necessary

to

transform

these

attached

silver

ions/salts

into

metallic

silver

particles

so

that

they

may

subsequently

work

as

nuclei

for

subsequent

growth

of

silver

particles

in

the

physical

developer

Because

the

reducing

agent

in

the

physical

developer

is

ready

to

reduce

these

attached

silver

ions/salts

into

metallic

silver

nuclei

an

isolated

step

for

reduction

is

not

necessary

before

the

treatment

in

the

physical

developer

However

a

brief

exposure

of

the

silver-impregnated

sections

to

formalin

prior

to

the

physical

development

leads

to

an

instantaneous

completion

of

the

physical

development

-LSB-

-RSB-

However

it

is

possible

that

silver

ions

contained

in

the

physical

developer

potentially

attaches

to

the

tissue

and

are

physically

developed

to

form

separate

metallic

silver

particles

Because

this

may

happen

independently

of

the

silver

from

the

initial

alkaline

silver

iodide

solution

-LSB-

-RSB-

a

prolonged

incubation

in

the

physical

developer

may

be

hampered

by

this

unexpected

staining

Therefore

pretreatments

have

been

invented

in

order

to

minimize

this

background

A

pretreatment

initially

proposed

by

Gallyas

was

periodic

acid

for

min

He

tried

various

pretreatments

-LSB-

-RSB-

and

finally

proposed

an

alternative

pretreatment

with

lanthanum

sodium

acetate

in

for

h

which

is

more

effective

in

eliminating

the

background

-LSB-

-RSB-

An

additional

pretreatment

with

sodium

permanganate

followed

by

oxalic

acid

-LSB-

-RSB-

-LRB-

initially

intended

for

melanin

bleaching

-LSB-

-RSB-

-RRB-

prior

to

this

lanthanum

nitrate/sodium

acetate

treatment

of

Gallyas

-LSB-

-RSB-

was

found

highly

effective

in

eliminating

the

background

and

is

currently

in

use

as

the

standard

in

our

laboratory

The

background

staining

is

practically

negligible

because

the

expected

target

staining

is

usually

obtained

before

long

and

this

untoward

background

reaction

is

generally

slow

Similar

oxidative

pretreatments

are

not

beneficial

for

other

silver-staining

methods

-LSB-

-RSB-

The

subsequent

gold

toning

is

essentially

similar

to

that

for

Bodian

method

Compared

with

Bodian

and

Bielschowsky

methods

Gallyas

method

is

characterized

by

its

stability

reproducibility

and

ability

to

visualize

abundant

deposits

-LSB-

-RSB-

Moreover

normal

structures

are

not

stained

in

practice

This

allows

easy

and

reproducible

examination

especially

when

looking

for

pathological

deposits

such

as

NFTs

or

glial

cytoplasmic

inclusions

-LRB-

GCIs

-RRB-

of

multiple

system

atrophy

-LRB-

MSA

-RRB-

-LSB-

-RSB-

However

amyloid

deposits

are

hardly

visualized

and

it

is

usually

neuritic

components

of

SPs

that

are

labeled

by

Gallyas

method

Campbell

Switzer

method

The

initial

impregnation

solution

of

Campbell

Switzer

method

contains

pyridine

-LRB-

Table

-RRB-

Pyridine

was

initially

used

for

tissue

fixation

Bielschowsky

-LSB-

-RSB-

used

pyridine

for

postfixation

of

formaldehyde-fixed

frozen

sections

and

found

an

enhanced

selectivity

In

order

to

avoid

instability

and

risk

of

explosion

of

the

ammoniacal

silver

solution

Hicks

-LSB-

-RSB-

added

pyridine

-LRB-

vol

-RRB-

to

the

silver

nitrate

solution

-LRB-

-RRB-

buffered

with

potassium

carbonate

at

alkaline

range

Thereafter

Campbell

et

al.

-LSB-

-RSB-

combined

this

silver-pyridine

-LRB-

vol

-RRB-

carbonate

solution

with

the

physical

developer

invented

by

Gallyas

and

found

that

this

staining

is

not

only

highly

sensitive

in

detecting

SPs

and

NFTs

in

AD

brains

but

also

as

stable

and

reproducible

as

Gallyas

method

The

solution

was

later

modified

by

increasing

the

concentration

of

pyridine

up

to

-LSB-

-RSB-

and

was

found

to

be

equally

very

powerful

in

detecting

α-synuclein-related

pathologies

-LSB-

-RSB-

Gallyas

demonstrated

that

the

presence

of

pyridine

markedly

decreases

the

amount

of

silver

either

metallic

or

reducible

attached

to

the

section

-LSB-

-RSB-

This

is

compatible

with

the

observation

that

pyridine

enhances

the

selectivity

of

Bielschowsky

method

-LSB-

-RSB-

and

that

a

higher

concentration

of

pyridine

is

associated

with

reduced

background

-LSB-

-RSB-

Because

the

standard

pretreatment

-LRB-

potassium

permanganate/oxalic

acid

followed

by

lanthanium

nitrate

-RRB-

for

Gallyas

method

does

not

affect

the

results

obtained

with

Campbell

Switzer

method

and

there

are

essentially

no

differences

between

the

physical

developers

used

for

Gallyas

and

Campbell

Switzer

methods

the

only

significant

difference

between

them

is

the

silver

solutions

-LSB-

-RSB-

The

reason

for

these

differences

between

Gallyas

and

Campbell

Switzer

methods

remains

unexplained

Enhancement

of

silver

staining

by

methenamine

Methenamine

has

been

used

for

histological

demonstration

of

glycogen

and

mucin

-LSB-

-RSB-

Periodic

acid-methenamine

silver

stain

has

been

used

for

staining

of

the

basement

membrane

of

the

glomeruli

-LSB-

-RSB-

Dekura

et

al.

-LSB-

-RSB-

first

described

the

use

of

the

methenamine-silver

solution

for

neurofibrils

without

prior

periodic

acid

Further

modification

of

this

methenamine-silver

method

was

found

highly

sensitive

in

detecting

SPs

which

is

equivalent

to

immunohistochemistry

for

amyloid

β-protein

-LRB-

Aβ

-RRB-

-LSB-

-RSB-

Interestingly

formic

acid

pretreatment

which

enhances

immunoreactivity

for

Aβ

of

SPs

completely

diminishes

argyrophilia

of

SPs

by

the

methenamine-silver

method

or

congophilia

suggesting

that

the

methenamine-silver

method

detects

specific

structures

such

as

β-pleated

sheets

of

amyloid

which

are

destroyed

by

formic

acid

-LSB-

-RSB-

It

also

stains

perivascular

amyloid

but

fails

to

stain

Kuru

plaque

of

Gerstmann

Sträussler

Scheinker

disease

-LSB-

-RSB-

and

renal

deposits

of

amyloid

-LSB-

-RSB-

This

is

in

agreement

with

the

early

speculation

by

Seki

-LSB-

-RSB-

that

factors

other

than

direct

affinity

of

silver

to

some

tissue

elements

such

as

the

quantity

of

intervening

colloid

or

packing

density

of

tissue

components

are

also

responsible

for

final

results

of

the

silver

staining

The

paucity

of

normal

neurofibrils

with

this

method

is

helpful

in

delineating

SPs

on

electron

microscopy

-LSB-

-RSB-

It

is

then

possible

to

detect

even

sparse

aggregations

of

amorphous

often

ramified

structures

with

fine

granular

silver

deposits

suggesting

that

this

highly

sensitive

method

is

capable

of

detecting

the

earliest

stage

of

Aβ

deposition

-LSB-

-RSB-

Although

the

mechanism

of

lesion-specific

enhancement

remains

unknown

introduction

of

of

methenamine

in

the

silver

proteinate

solution

of

Bodian

method

is

similarly

effective

in

enhancing

the

selective

affinity

to

SPs

and

NFTs

-LSB-

-RSB-

It

is

interesting

that

this

methenamine-silver

method

exhibits

more

affinity

to

Pick

bodies

when

they

are

extracellular

-LSB-

-RSB-

while

intracellular

counterparts

are

more

readily

stained

by

conventional

Bodian

method

even

with

electron

microscopy

-LSB-

-RSB-

Pretreatment

with

microwave

A

microwave

treatment

of

sections

in

silver

solution

was

first

reported

by

Brinn

-LSB-

-RSB-

This

treatment

causes

an

increase

in

molecular

movements

across

the

tissue

This

presumably

facilitates

quicker

silver

impregnation

At

the

same

time

shortened

incubation

time

for

impregnation

and

activated

molecular

movements

may

minimize

precipitate

formation

Microwave

activation

of

silver

impregnation

for

SPs

and

NFTs

was

first

introduced

in

order

to

apply

King

's

method

-LSB-

-RSB-

-LRB-

originally

designed

for

frozen

sections

-RRB-

to

paraffin-embedded

sections

-LSB-

-RSB-

Microwave

activation

is

effective

with

Bielschowsky

-LSB-

-RSB-

and

the

methenamine

silver

-LSB-

-RSB-

methods

in

shortening

the

incubation

time

for

silver

impregnation

and

eliminating

the

background

Although

the

elevated

temperature

may

partly

explain

the

improvements

of

these

silver-staining

methods

it

has

also

been

reported

that

a

lowered

temperature

at

°C

for

initial

impregnation

in

silver

nitrate

for

Yamamoto

Hirano

's

modification

of

Bielschowsky

method

gives

more

reproducible

results

in

visualizing

a

larger

number

of

diffuse

plaques

-LSB-

-RSB-

Molecular

species

detected

by

silver

staining

It

is

interesting

to

identify

isolated

molecular

species

detected

by

silver-staining

methods

but

these

attempts

have

been

so

far

limited

Gambetti

et

al.

used

Bodian

method

and

anti-neurofilament

antibodies

in

parallel

to

stain

an

extract

from

rat

spinal

cord

electrophoresed

on

the

SDS-polyacrylamide

gel

The

typical

triplet

of

and

kDa

identified

on

the

Bodian-stained

gel

is

in

agreement

with

the

neurofilament

triplet

identified

on

the

counterpart

stained

with

anti-neurofilament

antibodies

-LSB-

-RSB-

They

confirmed

similar

parallelism

in

other

vertebrate

and

nonvertebrate

species

as

well

-LSB-

-RSB-

Additional

less

intense

bands

at

a

lower

molecular

weight

range

may

correspond

to

tubulin

or

degradation

products

of

neurofilament

proteins

-LSB-

-RSB-

Because

these

findings

have

been

obtained

with

normal

nerve

tissue

further

analyses

based

on

brains

containing

pathological

lesions

are

mandatory

to

identify

components

responsible

for

their

argyrophilia

on

the

pathological

lesions

Iqbal

et

al.

-LSB-

-RSB-

used

Gallyas

method

to

stain

the

SDS-gel

after

electrophoresis

of

the

paired

helical

filament

-LRB-

PHF

-RRB-

fraction

extracted

from

AD

brain

The

electrophoretic

mobilities

of

the

Gallyas-positive

bands

correspond

to

those

of

PHF

tau

-LRB-

intense

-RRB-

normal

tau

-LRB-

less

intense

-RRB-

and

high

molecular

weight

MAPs

-LRB-

weak

-RRB-

The

absence

of

Gallyas-positive

bands

in

the

range

of

neurofilament

proteins

is

in

sharp

contrast

with

the

Bodian-positive

triplet

of

neurofilament

-LSB-

-RSB-

This

difference

may

explain

why

Gallyas

method

is

practically

free

from

the

staining

of

normal

structures

such

as

neuronal

soma

and

neurites

This

discrepancy

suggests

that

molecular

species

detected

by

different

silver-staining

methods

can

be

completely

different

even

though

both

of

the

methods

label

apparently

identical

structures

such

as

NFTs

on

histological

sections

Because

several

silver-staining

methods

have

been

modified

to

stain

SDS

gels

-LSB-

-RSB-

it

may

be

possible

to

identify

molecular

species

either

normal

or

specific

to

some

pathological

structures

which

have

a

selective

affinity

to

some

silver-staining

methods

Because

extracted

proteins

electrophoresed

on

gel

are

necessarily

modified

or

significantly

degenerated

this

approach

may

be

fruitful

only

if

the

disease-specific

nature

if

detectable

with

silver-staining

methods

is

readily

represented

by

a

single

isolated

molecule

even

when

it

is

modified

for

electrophoresis

Quantitative

comparison

of

silver-staining

methods

and

immunohistochemistry

Even

after

the

introduction

of

immunohistochemistry

modification

of

classic

silver-staining

methods

or

invention

of

new

silver-staining

methods

have

been

attempted

-LSB-

-RSB-

Two

major

deposits

targeted

by

these

silver-staining

methods

are

undoubtedly

NFTs

and

SPs

of

AD

-LSB-

-RSB-

Different

silver-staining

methods

-LSB-

-RSB-

have

been

compared

quantitatively

for

their

relative

ability

to

detect

NFTs

or

SPs

as

represented

in

Fig.

-LSB-

-RSB-

In

respect

to

detection

of

NFTs

these

comparative

studies

are

in

agreement

that

Gallyas

method

is

most

powerful

followed

by

one

of

the

three

methods

modified

Bielschowsky

Bodian

or

Cross

methods

The

methenamine-silver

method

is

rated

as

least

powerful

for

NFTs

There

are

two

studies

each

claiming

a

superior

or

equivalent

number

of

NFTs

detected

with

Reusche

method

-LSB-

-RSB-

or

with

the

nickel

peroxidase

method

-LSB-

-RSB-

compared

with

Gallyas

method

This

rating

of

putative

sensitivity

however

is

not

shared

when

SPs

are

examined

SPs

are

well

stained

by

modified

Bielschowsky

-LSB-

-RSB-

the

methenamine-silver

-LSB-

-RSB-

periodic

acid

methenamine

silver

-LSB-

-RSB-

or

thioflavin-S

-LSB-

-RSB-

to

similar

extent

Bodian

method

is

less

powerful

in

detecting

SPs

especially

diffuse

deposits

-LSB-

-RSB-

Gallyas

Cross

-LSB-

-RSB-

and

Garvey

-LSB-

-RSB-

methods

are

least

powerful

in

detecting

SPs

Fig.

of

NFTs

-LRB-

a

upper

panel

-RRB-

and

that

of

SPs

-LRB-

b

lower

panel

-RRB-

as

a

function

of

intellectual

status

evaluated

by

the

Blessed

Test

Score

Both

are

dependent

on

the

staining

methods

Reproduced

with

permission

-LSB-

-RSB-

It

is

needless

to

say

that

a

more

sensitive

detection

is

preferable

in

order

to

identify

specific

deposits

in

more

detail

with

ease

However

if

one

deals

with

clinicopathological

correlation

of

a

disease

such

as

AD

where

symptoms

and

deposits

are

more

or

less

quantifiable

under

the

background

of

normal

aging

it

is

possible

to

identify

a

threshold

of

the

deposits

with

any

of

the

methods

that

may

correlate

with

a

threshold

of

the

cognitive

decline

-LSB-

-RSB-

For

this

purpose

stability

and

reproducibility

of

the

method

are

preferable

-LSB-

-RSB-

Reliable

agreement

between

the

conventional

Braak

staging

of

neurofibrillary

pathology

based

on

Gallyas

method

-LSB-

-RSB-

and

the

revised

protocol

based

on

immunohistochemistry

-LSB-

-RSB-

is

a

good

example

On

the

other

hand

if

one

is

looking

for

specific

pathological

deposits

such

as

Pick

bodies

GCIs

or

astrocytic

plaques

not

observed

in

normal

aging

-LSB-

-RSB-

specificity

is

the

primary

concern

for

the

staining

method

Some

of

these

comparative

studies

included

immunohistochemistry

for

tau

-LSB-

-RSB-

Aβ

-LSB-

-RSB-

ubiquitin

-LSB-

-RSB-

or

neurofilament

-LSB-

-RSB-

for

comparison

with

silver-staining

methods

Some

studies

with

highly

sensitive

Aβ

immunohistochemistry

demonstrated

that

it

visualizes

a

larger

number

of

deposits

than

with

conventional

silver-staining

methods

-LSB-

-RSB-

This

suggests

that

amorphous

Aβ

deposition

not

identified

by

conventional

silver-staining

methods

represents

the

earliest

stage

of

Aβ

deposition

-LSB-

-RSB-

On

the

other

hand

it

is

noteworthy

that

of

the

series

found

any

quantitative

superiority

of

immunohistochemistry

in

identifying

NFTs

over

the

best-possible

silver-staining

method

For

example

it

is

rather

frequent

that

pathological

structures

in

the

form

of

NFT

do

not

exhibit

tau-like

immunoreactivity

any

more

while

most

of

them

are

stained

by

some

silver-staining

methods

or

fluorochromes

-LSB-

-RSB-

This

is

explained

if

some

tau

epitopes

in

NFTs

are

truncated

out

during

their

evolution

from

pretangle

to

extracellular

NFTs

-LSB-

-RSB-

Furthermore

immunohistochemical

detection

is

highly

dependent

on

the

antibody

and

experimental

procedures

These

technical

uncertainties

make

it

difficult

to

place

immunohistochemical

detection

as

a

standard

for

neuropathological

diagnosis

especially

when

the

quantity

of

deposits

is

of

primary

importance

as

with

the

histological

diagnostic

criteria

for

AD

-LSB-

-RSB-

It

is

needless

to

say

that

the

molecular

specificity

of

immunohistochemistry

is

a

potentially

reliable

and

logical

way

in

order

to

sort

different

diseases

under

the

diagnostic

flags

of

molecules

This

highly

specific

detection

by

pinpointing

a

single

target

molecule

is

however

a

very

restricted

monochromic

view

as

pointed

out

by

Switzer

-LSB-

-RSB-

Identification

of

the

disease-specific

molecular

species

and

their

reliable

probes

applicable

to

histological

sections

are

desirable

Even

if

this

kind

of

probes

are

available

immunohistochemical

procedures

are

also

difficult

to

standardize

and

much

more

costly

than

most

of

silver-staining

methods

-LSB-

-RSB-

Immunohistochemistry

is

highly

costly

especially

when

staining

large

histological

samples

for

example

hemispheric

sections

of

human

brain

Moreover

penetration

of

antibodies

is

sometimes

not

sufficient

when

staining

thick

histological

sections

Silver-staining

methods

are

more

readily

applicable

even

to

large

and

thick

histological

sections

-LSB-

-RSB-

It

is

of

note

as

well

that

archival

materials

preserved

for

a

long

period

especially

in

formaldehyde

sometimes

fail

to

exhibit

immunoreactivity

It

is

frequently

possible

to

visualize

the

lesions

with

silver-staining

methods

even

in

these

archival

materials

-LSB-

-RSB-

Qualitative

representation

by

silver-staining

methods

As

summarized

in

the

previous

section

differences

in

the

quantity

of

argyrophilic

AD

lesions

are

dependent

on

the

silver-staining

method

As

has

been

considered

frequently

it

may

be

the

sensitivity

of

a

staining

method

that

governs

the

quantity

of

the

argyrophilic

lesions

This

is

probably

in

agreement

with

our

general

impression

that

modified

Bielschowsky

method

is

more

powerful

than

Bodian

method

in

detecting

AD

lesions

-LSB-

-RSB-

This

interpretation

is

based

on

the

assumption

that

so-called

argyrophilia

is

a

homogeneous

phenomenon

regardless

of

the

staining

method

and

the

nature

of

lesion

which

is

not

the

case

For

example

Gallyas

method

is

one

of

the

most

sensitive

methods

in

detecting

NFTs

and

neuropil

threads

in

AD

while

it

is

far

less

sensitive

in

detecting

SPs

This

empirical

but

evident

discrepancy

is

not

readily

explained

by

a

mere

difference

in

the

sensitivity

of

the

methods

and

rather

indicates

that

argyrophilic

properties

are

dependent

not

only

on

the

staining

methods

but

also

on

the

target

lesions

Careful

neuropathologists

have

been

aware

of

these

method

and

lesion-dependent

natures

of

various

argyrophilic

deposits

in

neurodegenerative

diseases

-LSB-

-RSB-

Table

provides

a

summary

of

the

heterogeneity

of

argyrophilia

according

to

the

staining

method

and

the

lesion

Gallyas

method

is

one

of

the

most

sensitive

silver-staining

method

that

clearly

labels

NFTs

of

AD

as

well

as

neuronal

and

glial

lesions

of

corticobasal

degeneration/progressive

supranuclear

palsy

-LRB-

CBD/PSP

-RRB-

-LSB-

-RSB-

This

sensitive

method

however

fails

to

stain

Pick

bodies

another

distinct

tau-positive

deposit

-LSB-

-RSB-

On

the

other

hand

Campbell

Switzer

method

labels

Pick

bodies

but

not

CBD/PSP-related

lesions

-LSB-

-RSB-

This

reversed

and

complementary

profile

with

Gallyas

and

Campbell

Switzer

methods

is

hardly

explained

by

a

superior

sensitivity

of

Gallyas

method

over

Campbell

Switzer

method

or

vise

versa

These

discrepancies

between

Gallyas

and

Campbell

Switzer

methods

are

readily

demonstrable

if

mirror

section

pairs

are

initially

fluorolabeled

and

one

of

the

section

pair

is

subsequently

stained

with

Gallyas

and

the

counterpart

with

Campbell

Switzer

method

as

shown

in

Fig.

It

is

now

known

that

molecular

species

of

tau

are

different

according

to

the

disease-specific

lesions

Namely

Pick

bodies

are

usually

composed

of

three-repeat

-LRB-

-RRB-

tau

while

CBD/PSP-related

tau

pathologies

are

positive

for

four-repeat

-LRB-

-RRB-

tau

-LSB-

-RSB-

Because

Pick

bodies

are

usually

positive

with

Campbell

Switzer

method

but

negative

with

Gallyas

method

-LRB-

Fig.

d

-RRB-

one

of

the

possible

interpretations

is

that

tau

deposits

such

as

Pick

bodies

exhibit

argyrophilia

with

Campbell

Switzer

but

not

with

Gallyas

method

In

contrast

the

lesions

of

tauopathy

-LRB-

CBD/PSP

and

argyrophilic

grain

disease

-LSB-

-RSB-

-RRB-

are

related

to

the

argyrophilia

with

Gallyas

but

not

with

Campbell

Switzer

method

-LRB-

Fig.

l

-RRB-

-LSB-

-RSB-

Indeed

NFTs

of

AD

Down

syndrome

and

diffuse

neurofibrillary

tangles

with

calcification

all

containing

both

and

isoforms

similarly

exhibit

argyrophilia

with

either

method

-LRB-

Fig.

h

-RRB-

-LSB-

-RSB-

It

is

interesting

that

Gallyas-positive

Pick-like

bodies

were

found

to

be

positive

for

tau

but

sparsely

positive

for

tau

-LSB-

-RSB-

which

corroborates

the

empirical

correlation

between

tau

and

Gallyas

method

Moreover

in

one

of

the

largest

studies

on

sporadic

and

familial

frontotemporal

degeneration

with

tau-positive

deposits

Pick

bodies

defined

as

inclusions

stained

by

Bielschowsky

but

not

by

Gallyas

were

found

in

cases

All

exhibited

the

immunoreactivity

with

a

specific

antibody

as

well

as

tau

predominance

in

tau

biochemistry

except

for

a

single

case

with

mutation

in

the

tau

gene

-LSB-

-RSB-

This

particular

case

is

characterized

by

diffuse

cytoplasmic

tau

staining

in

neurons

and

PHF-like

electron

microscopic

features

-LSB-

-RSB-

both

of

which

are

not

typical

of

Pick

bodies

In

another

sporadic

case

Bodian-positive

inclusions

positive

for

but

not

tau

have

electron

microscopic

features

with

twisted

ribbon

distinct

from

those

of

typical

Pick

bodies

-LSB-

-RSB-

These

apparent

heterogeneities

necessitate

a

revision

of

the

classical

definition

of

Pick

bodies

round

argyrophilic

tau-positive

inclusions

in

neuronal

cytoplasm

because

argyrophilic

features

and

tau-species

are

both

heterogeneous

in

this

classical

category

Among

argryophilic

cytoplasmic

inclusions

in

neuron

it

seems

that

those

positive

for

-LSB-

-RSB-

and

those

negative

with

Gallyas

method

-LSB-

-RSB-

are

correlated

to

form

a

cluster

to

be

defined

as

Pick

bodies

Argyrophilia

with

the

Campbell

Switzer

method

-LSB-

-RSB-

is

helpful

for

more

precise

and

straightforward

definition

of

Pick

body

In

contrast

with

Gallyas

and

Campbell

Switzer

methods

argryrophilia

with

Bielschowsky

and

Bodian

methods

are

less

dependent

on

the

target

lesions

-LRB-

Table

-RRB-

It

is

noteworthy

that

tau-positive

cortical

lesions

of

PSP

are

silver-stained

with

Bodian

method

-LSB-

-RSB-

while

those

of

CBD

frequently

fail

to

exhibit

argyrophilia

with

Bodian

method

-LSB-

-RSB-

especially

in

the

cerebral

cortices

Although

possible

biochemical

distinctions

of

tau

between

CBD

and

PSP

have

been

described

-LSB-

-RSB-

it

remains

to

be

clarified

how

these

biochemical

differences

are

related

to

these

morphological

differences

between

CBD

and

PSP

Another

possible

limitation

is

related

to

the

observations

that

the

expression

of

tau-isoforms

is

not

homogeneous

throughout

the

brain

-LSB-

-RSB-

Because

most

of

the

data

on

tau-positive

deposits

summarized

in

Table

and

Fig.

have

been

obtained

on

cerebral

cortices

it

remains

to

be

determined

whether

these

findings

are

similarly

applicable

to

extracortical

areas

such

as

the

brainstem

Yoshida

-LSB-

-RSB-

demonstrated

that

tau

immunoreactivity

is

represented

in

the

brainstem

even

in

so-called

tauopathies

such

as

CBD/PSP

Because

neurofibrillary

pathologies

related

to

AD

or

aging

are

sometimes

found

in

the

brainstem

nuclei

and

usually

positive

for

both

and

isoforms

it

remains

to

be

clarified

whether

the

presence

of

tau

in

brainstem

nuclei

in

CBD/PSP

is

ascribed

to

tauopathies

independent

of

normal

or

accelerated

aging

Table

of

argyrophilia

according

to

disease

and

methodStaining

methodsCampbell

SwitzerBodianBielschowskyGallyasPick

bodies

-LSB-

-RSB-

AD-NFTs

Down-NFTs

DNTC-NFTs

PSP/CBD-neurons

a

b

PSP/CBD-glia

c

b

Argyrophilic

grains

-LSB-

-RSB-

AD-SPs

AD-diffuse

deposits

Lewy

bodies

-LSB-

-RSB-

GCIs

-LSB-

-RSB-

AD

Alzheimer

's

disease

NFTs

neurofibrillary

tangles

DNTC

diffuse

NFTs

with

calcification

PSP

progressive

supranuclear

palsy

CBD

corticobasal

degeneration

SP

senile

plaques

GCIs

glial

cytoplasmic

inclusions

easily

recognizable

positive

questionably

positive

negativeaPositive

in

PSP

cases

-LSB-

-RSB-

but

less

evident

in

CBD

cases

-LSB-

-RSB-

bNot

consistent

-LSB-

-RSB-

positive

in

some

cases

not

appropriate

for

histological

examinationcThe

argyrophilia

-LRB-

Bodian

-RRB-

is

less

evident

in

glia

than

in

neurons

especially

in

CBD

casesFig

profiles

of

various

deposits

in

neurodegenerative

diseases

compared

with

immunofluoresence

images

-LRB-

a

d

Pick

bodies

-LSB-

-RSB-

e

h

neurofibrillary

tangles

i

l

argyrophilic

grains

-LSB-

-RSB-

m

p

glial

cytoplasmic

inclusions

q

t

Lewy

bodies

-LSB-

-RSB-

-RRB-

Mirror

section

pairs

were

initially

multi-fluorolabeled

for

PHF-tau

-LRB-

green

for

b

c

f

g

j

k

-RRB-

or

for

α-synuclein

-LRB-

green

for

n

o

r

s

-RRB-

with

thiazin

red

-LRB-

red

fluorochrome

that

labels

fibrillary

structure

such

as

neurofibrillary

tangles

for

b

c

f

g

j

k

n

o

r

s

-RRB-

and

for

ubiquitin

-LRB-

blue

for

f

g

j

k

n

o

r

s

-RRB-

After

recording

the

fluorescent

images

from

the

same

area

of

the

mirror

section

pairs

one

of

the

section

pair

was

stained

with

Campbell

Switzer

-LRB-

CS

a

e

i

m

q

-RRB-

and

the

other

with

Gallyas

-LRB-

GAL

d

h

l

p

t

-RRB-

method

Fluorescent

images

and

silver

staining

profiles

of

the

same

area

from

the

mirror

section

pair

were

compared

Pick

bodies

-LRB-

a

d

-RRB-

in

the

pyramidal

layer

of

hippocampus

are

stained

with

CS

-LRB-

a

-RRB-

but

not

with

GAL

-LRB-

d

-RRB-

-LSB-

-RSB-

Neurofibrillary

tangles

-LRB-

NFTs

e

h

-RRB-

from

the

frontal

cortex

of

diffuse

neurofibrillary

tangles

with

calcification

are

stained

with

both

CS

-LRB-

e

-RRB-

and

GAL

-LRB-

h

-RRB-

-LSB-

-RSB-

This

staining

profile

is

shared

with

NFTs

of

AD

-LSB-

-RSB-

Argyrophilic

grains

-LRB-

i

l

in

the

square

-RRB-

and

tau-positive

neurons

are

present

-LRB-

j

k

-RRB-

in

the

parahippocampal

gyrus

The

tau-positive

neuron

-LRB-

double

asterisk

-RRB-

and

neuropil

thread

-LRB-

arrow

-RRB-

stained

with

both

CS

and

GAL

are

similar

to

those

observed

in

DNTC

-LRB-

e

h

-RRB-

representing

neurofibrillary

pathology

Grains

and

pretangle

neuron

-LRB-

single

asterisk

-RRB-

are

stained

with

GAL

-LRB-

l

-RRB-

but

not

with

CS

-LRB-

i

-RRB-

in

contrast

This

staining

profile

is

shared

with

cortical

lesion

of

corticobasal

degeneration/progressive

supranuclear

palsy

-LSB-

-RSB-

Glial

cytoplasmic

inclusions

-LRB-

m

p

-RRB-

in

the

putamen

positive

for

α-synuclein

-LRB-

n

o

-RRB-

are

stained

with

both

CS

-LRB-

m

-RRB-

and

GAL

-LRB-

p

-RRB-

-LSB-

-RSB-

Lewy

bodies

-LRB-

arrowhead

q

t

-RRB-

and

Lewy

neurites

-LRB-

arrow

q

t

-RRB-

in

the

dorsal

motor

nucleus

of

vagus

positive

for

α-synuclein

-LRB-

r

s

-RRB-

are

stained

with

CS

-LRB-

q

-RRB-

but

not

with

GAL

-LRB-

t

-RRB-

-LSB-

-RSB-

The

same

blood

vessel

is

indicated

with

asterisk

-LRB-

q

t

-RRB-

Bar

μm

-LRB-

a

d

m

p

q

t

-RRB-

bar

μm

-LRB-

e

l

-RRB-

Reproduced

with

permission

a

d

-LSB-

-RSB-

e

l

-LSB-

-RSB-

m

t

-LSB-

-RSB-

Interestingly

argyrophilic

differentiation

with

Gallyas

and

Campbell

Switzer

methods

is

not

limited

to

tau-positive

deposits

Both

of

the

two

staining

methods

label

GCIs

of

MSA

-LRB-

Fig.

p

-RRB-

-LSB-

-RSB-

Although

GCIs

are

positive

for

some

restricted

tau

epitopes

-LSB-

-RSB-

their

major

constituent

is

now

considered

to

be

α-synuclein

Indeed

Campbell

Switzer

method

clearly

labels

Lewy

bodies

and

their

neurites

another

representative

deposits

of

α-synuclein

-LSB-

-RSB-

It

is

interesting

that

Gallyas

method

fails

to

label

Lewy

bodies

and

Lewy

neurites

-LRB-

Fig.

t

-RRB-

Therefore

these

two

silver-staining

methods

when

performed

in

parallel

provide

staining

profiles

that

may

allow

qualitative

distinction

between

GCIs

and

Lewy

bodies

-LSB-

-RSB-

Because

no

biochemical

markers

that

enable

distinction

between

Lewy

bodies

and

GCIs

are

not

readily

available

these

differential

profiles

in

silver

staining

provides

an

additional

qualitative

distinction

possibly

linked

to

underlying

molecular

differences

At

present

these

empirical

distinctions

are

useful

for

diagnostic

differentiation

but

await

further

explanations

preferably

at

molecular

or

ultrastructural

level

These

distinctive

features

of

silver-staining

profiles

may

be

explained

if

-LRB-

-RRB-

molecular

species

specific

to

each

subcategory

-LRB-

for

example

vs.

tau

or

LBs

vs.

GCIs

-RRB-

has

different

affinity

to

each

silver-staining

method

-LRB-

-RRB-

some

disease-specific

ultrastructures

of

pathological

deposit

are

related

to

each

staining

profile

-LRB-

-RRB-

other

independent

molecules

in

these

deposits

play

some

roles

in

characterizing

the

pathological

deposits

so

that

distinctive

silver-staining

profiles

are

engendered

or

-LRB-

-RRB-

some

disease-specific

conformational

changes

are

related

to

a

silver-staining

profile

specific

to

a

disease

Because

Campbell

Switzer

and

Gallyas

methods

are

useful

in

discriminating

not

only

tau-positive

deposits

but

also

α-synuclein-positive

deposits

even

with

their

molecular

and

ultrastructural

diversities

it

is

plausible

that

factors

such

as

-LRB-

-RRB-

or

-LRB-

-RRB-

shared

by

tau-positive

deposits

and

synuclein-positive

deposits

may

play

roles

at

least

partly

in

these

discriminating

abilities

with

these

silver-staining

methods

Conclusions

and

perspective

Even

with

my

limited

experience

on

these

silver-staining

methods

it

is

apparent

that

the

argyrophilic

profiles

are

dependent

not

only

on

the

silver-staining

method

but

also

on

the

target

lesion

This

indicates

that

argyrophilia

is

highly

heterogeneous

However

this

heterogeneity

is

not

capricious

but

rather

representative

of

some

disease-specific

aspects

The

apparent

empirical

correlations

between

the

argyrophilic

profiles

and

the

disease-specific

pathological

structures

are

useful

in

sorting

these

neurodegenerative

disorders

Combined

silver-staining

profiles

for

example

with

Gallyas

and

Campbell

Switzer

methods

are

more

convenient

than

modified

Bielschowsky

and

Bodian

method

for

sorting

different

disease-specific

lesions

Although

these

staining

profiles

are

discussed

in

this

review

as

if

disease-specific

more

detailed

comparison

at

cellular

level

demonstrated

that

they

can

be

different

from

cell

to

cell

even

in

the

same

section

-LRB-

intercellular

differences

-RRB-

-LSB-

-RSB-

Furthermore

staining

profiles

are

heterogeneous

even

in

a

single

cell

-LRB-

intracellular

differences

-RRB-

-LSB-

-RSB-

These

silver-staining

and

immunohistochemical

profiles

are

much

more

informative

especially

when

combined

together

than

when

used

in

isolation

because

such

combination

is

not

a

mere

summation

of

the

results

obtained

with

different

methods

It

will

rather

provide

a

more

stereoscopic

and

multi-faceted

view

which

may

allow

more

precise

morphological

delineation

of

disease-specific

deposits

In

my

view

comments

such

as

Silver-staining

methods

are

unreliable

nonreproducible

nonscientific

and

out-dated

are

now

out-dated

Awareness

of

their

utility

and

reliability

of

silver-staining

methods

different

from

immunohistochemistry

may

facilitate

diagnosis

and

accelerate

research

which

hopefully

clarifies

molecular

basis

of

each

silver-staining

method

and

improves

our

understanding

of

diseases

argyrophilia
gallyas
tauopathies
senile plaques
methenamine