Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/

Myofibrillar ATPase histochemistry of rat skeletal muscles: a "two-dimensional" quantitative approach. A Lind and D Kernell J Histochem Cytochem 1991 39: 589 DOI: 10.1177/39.5.1826695 The online version of this article can be found at: http://jhc.sagepub.com/content/39/5/589

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The Journal of Histochemistry and Cytochemistry Copyright © 1991 by The Histochemical Society, Inc.

Vol.

and

ALBERT

LIND

Department

ofNeurophysiology,

Received

for

publication

9,

1990

and

ofAmsterdam, in revised

form

November

study, we have been exploring approach for the quadripartite

a quantitative histochemical

“twoclas-

sification of muscle fibers in rat limb muscles. We were motivated to perform this analysis as a background for the use of rats rather cats in further

of neuromuscular In histochemical are usually

classified

ing for myofibrillar employed largely

techniques by utilizing

physiological

and

histochemical

investigations

organization and properties. studies of skeletal muscles, into

three

ATPase

types

(mATPase).

causes the

main

One

a differential

varying

the various

according ofthe

most

staining

sensitivities

was shown justment sification

perimental

was originally by Brooke

and

primarily Kaiser

themselves

of the pH of the pre-incubation technique

animals

was

applicable

as well (1,2).

that,

fibers

mATPase

suitable

the same of common

have

to

method” (BK IIB. Although

after

medium, studies

commonly

for use in human,

to muscles

Other

stain-

ofmuscle

of their

developed

fibers

to their

acid or alkaline pH (1); in this “Brooke and Kaiser method) the fiber types are referred to as I, IIA, and

this method

Medisch 29,

it adclascx-

demonstrated

Centrum,

1990;

Muscles:

1105

accepted

AZ

December

Amsterdam, 4,

1990

The

Netherlands.

(0A2034).

the

myosin

highly

and

plastic,

instance,

mATPase

varying

the

age

with

of the

composition muscle

animals

of skeletal

usage

(20)

at

(10,11,23);

in this

fiber

are referred

types

GS methods lent staining

alkaline

pH

“Guth

with and

1

Correspondence

sterdam,

to: D. Kernell,

Academisch Medisch The Netherlands.

Dept.

Centrum,

of Neurophysiology, Meibergdreefls,

Univ. of 1105 AZ Am-

is

as with,

for

(14,16).

In addition to the BK method, another nique also produces a tripartite classification treatment

muscles

as well

frequently used by combining

techpre-

the

influence

of formaldehyde

Samaha

method”

(GS

to as a, a3, and

method)

Although

.

the

the

BK and

have commonly been considered to produce equivapatterns (i.e., I = 13, IIA = a, IIB = c43; if. 1,11,2 3),

this is not strictly

have revealed

true;

particularly

considerable

in rats,

variability

systematic

comparisons

with respect

to the relations

between fibers ofthe hA and IIB categories (BK method) and those ofthe a and c43 types (GS method) (9). Such a lack ofprecise correspondence ply

be

between

interpreted

as a kind

the two mATPase as a sign

of undesirable

spondence

might

differences

between

instance,

indicate

“noise.”

carry

techniques

of methodological

specific

(5) might

histochemical muscle,

referred

Alternatively,

the

information

of its own;

to two staining

methods

the presence

of systematic

differences

like hA fibers

to as Type

of a new 2X, whose

by the GS method

category

mATPase

i.e.,

lack

reactions

demonstration

sim-

imperfection,

ase properties that were not detected by one technique latter possibility is strongly suggested by the recent Amsterdam,

1991

USA.

separated by theirstaining densities for fixed allc-AThase (IIBd dark, IIBm moderate). The mean intensity ofSDH staining per fiber type, as measured in the central core ofthe fibers, was ranked such that IIA>I>IIBd>llBm. The analyzed musdes (tibialis anterior, biceps brachii) were markedly heterogeneous with respect to the topographic distribution of different fiber types. In comparison to other musde portions, the regions containing Type I fibers (“red” portions) showed a higher IIBd vs IIBm ratio and more intense SDH staining for either subtype of the IIB fibers. The llBd fibers probably correspond to the Type 2X fibers ofSchiaffmo et al. (J Histochem Cytochem 39:589-597, 1991) KEY WORDS: Rat; Skeletal muscle; Quantitative histochemistry; Myofibrillar ATPase; Succinate dehydrogenase; Fiber types.

that

Introduction

than

of Rat Skeletal Approach

Academisch

In histochemical investigations ofskeletal musde, the fibers are commonly dassiuied into three types according to their staining for myofibrillar ATPase (mATPase). In serial sections of skeletal musdes from normal Wistar rats, we compared two common staining methods for mATPase: (a) an ac-ATPase technique, with pre-incubation at pH 4.7, and (b) a fixed alk-ATPase technique, using treatment with 5% paraformaldehyde followed by pre-incubation at pH 10.4. In addition, the same fibers were stained in subsequent serial sections for succinate dehydrogenase (SDH) activity. Staining intensities were objectively evaluated by microphotomettic measurements ofoptical density. Combining both mATh a.se methods in consecutive serial sections (“two-dimensional approach”) led to the identification offour distinct dusters of fibers: Types I, HA, and two subgroups of Type HB, as

In the present dimensional”

589-597,

KERNELI)

University

July

pp.

5,

Article

Histochemistry Quantitative

DANIEL

No.

Pr:nted:n

Original

Myofibrillar ATPase A “Two-dimensional”

39,

of correconsistent might,

alone. The immuno-

of myosin was found

and like IIB fibers

for

in mATh

in rat to stain

by the BK 589

Downloaded from jhc.sagepub.com at SWINBURNE UNIV on September 2, 2014

590

LIND,

method (24,25; ing new fiber In the parison

for other recent immunohistochemical types, see 14,16).

present between

investigation,

we have

the

reactions

mATPase

studies

made

a systematic

of muscle

subjective

judgements

absorption

with simple

quantitative

approach

tween

the present

ofstaining represents

analysis

and

ase classification

of muscle

Our results

demonstrated

rat, the combined to a categorization different IIB, here

different

enzyme

histochemical described have

of the major

preceding

been

This

differences

be-

studies

that,

for mixed

muscles

subdivisions of Type groups belonging to

different

dehydrogenase; Some

published

of the

techniques led into one of four

were also distinctly

(24,25).

vs IIB discrimination

by Brooke

corresponded

of the present

in abstract

form

in ox-

SDH).

The

to those observations

(15).

214.03)

Methods

All muscles were taken from normal young-adult account of the present study concerns tibialis biceps brachii (BB, n = 2) muscles, and these rats with weights of 265-365 g (ages about 3-4 measurements were made on other muscles from toid,

gastrocnemius

medialis,

extensor

Wistar rats. The detailed anterior (Tk, n = 3) and samples came from male months).

Supplementary

male and frmale

digitorum

longus).

sections,

the

glass

slides

were

freshly

coated

with

0.1%

Staining

Ac-ATPase.

This

Unfixed

sections

in a buffer

consisting

method

was similar

to the

were pre-incubated of 100 mM

one

at room

potassium

of Brooke

temperature

chloride

and

Kaiser

for 10 mm

in 100 mM

sodium

acetate, adjusted to pH 4.7 with acetic acid (22). Thereafter, the following consecutive steps were carried out (17): (a) sections washed for 30 sec in a 20 mM glycine buffer (pH 9.4) containing 20 mM CaCI2; (b) 25 mm incubation at room temperature in 40 mM glycine buffer (pH 9.4) contaming 20 mM CaCI2 and 2.5 mM ATP disodium salt; (c) sections washed in three 30-sec changes of 1% CaCl2; (d) sections kept in 2% CaCI2 for 3 mm; (e) sections washed in three 30-sec changes of distilled water; (f) sections

immersed

washed

in distilled

in 1% yellow

water;

ammonium

(h) sections

the sections

sulfide

embedded

for 30 sec; (g) sections

in glycerol

in a 5%

because

and

11.5%

it preserves After

cacodylate

sucrose.

enzyme

fixation,

and

methanol-free

(sw

Methanol-

activity

the sections

better

were

sub-

were

treated

as described

above

(Steps

c-h)

for ac-ATPase.

SDH. The incubation medium was prepared according to Reichmann and Pette (22): 100 mM phosphate buffer (pH 7.6), 5 mM ethylenediaminotetraacetic acid (EDTA), 1 mM potassium cyanide, 0.2 mM methylphenazine methosulfate, 50 mM succinate, and 1.5 mM nitroblue tetrazoAfter

a 12-mm water,

incubation

fixed

at 37”C,

for 30 mm

the sections

in 10%

1-mm changes of distilled in glycerol jelly.

neutral

were

briefly

washed

formalin,

and

washed

water. Thereafter,

the sections

were em-

Measurements The results

jelly.

were analyzed

(6,13,27).

The

staining

methods.

same

using

individual This

measurements

fibers

were

of relative

studied

for each

for two regions

was done

optical

density

one ofthe

of 100 fibers

from

three each

(see Results for further

details). All portions ofthe sections utilized were free from tissue disruption and freeze damage. Measurements of light transmittance were obtained using a microscope equipped with aZeiss photometer SF, coupled to a digital display and provided with a stabilized source muscle

Procedures

In the present context, an adequate pH control was of crucial importance. All pH measurements were obtained with a digital instrument providing readings of 0.01 pH units, and the correct functioning of the buffers utilized was controlled by measuring the pH of media at the onset as well as at the end ofa (pre)incubation period. In the case ofthe mATPase procedures, pH changes were less than 0.01 pH units during pre-incubation, and 0.03 units or less during the final incubation at pH 9.4.

(1).

to the one of Guth

5 mm

with 0.15 M sodium

(12).

reported

Sec.

for SDH were processed on the same day. Other consecutive sections were stored overnight at - 80’C and stained by two different methods for myofibrillar ATPase on the next day.

and

originally

(1).

jected to the following consecutive actions: (a) washing for 1 mm in 0.1 M Tris buffer(pH 7.8)containing 18 mM CaCl2; (b)pre-incubation at room temperature for 15 mm in a 20 mM glycine buffer (pH 10.4) containing 20 mM CaCl2; (c) washing in two 1-mm changes of 0.1 M Iris buffer (pH 7.8) containing 18 mM CaCI2; (d) incubation at 37”C for 25 mm in a freshly prepared medium (4)consisting of8 ml of 1.0 M Tris(hydroxy-methyl) aminomethane, 4 ml ofo.18 M CaCl2, and 60 mg ATP disodium salt. This medium was diluted to 30 ml with distilled water, adjusted to pH 9.4 with 0.1 N HCI, and finally diluted to 40 ml with distilled water. After incuba-

in three bedded

tions to be stained

Incubation

for

1% CaCl2

used

range

was similar

buffered

was

the

4.5-4.7)

at 4’C

containing formalin

case,

poly-i.-lysine.

solution 7.6 and

the conventional

hum. the

fixed

than

in distilled

In each

were

free paraformaldehyde

rats (del.

dissected muscle was kept slightly stretched while being frozen in isopentane cooled by liquid nitrogen. Serial sections of 10 tm were cut in a cryostat at - 20”C and mounted on glass slides. To prevent loss or wrinkling of the

with

This method

Sections

at pH

overlapped

for the rat (pH

Alk-ATPase. (10).

paraformaldehyde

tion,

and

Materials

(4.7)

and Kaiser

Fixed

on the mATh

limb

mATPase staining all muscle fibers

of our IIBd fibers

for 2X fibers

previously

light

(7,27).

In addition to the pre-incubation at pH 4.7, as described above, we routinely subjected serial sections to the same staining procedures but with pre-incubation at other acidic pH values (always 4.6 and 4.8; sometimes varying in 0.1 unit steps from 4.0 to 4.9). Analysis of these various sections generally showed that, for the muscles of the present study, pre-incubation at pH 4.7 produced the best separation ofstaining intensities into the three Types I, IIA, and IIB (1) (Figures 1 and 2). Our optimal pH value for IIA

Samaha

(succinate

properties

In order associated

fibers.

categories activity

when

we measured

techniques

Type I, Type IIA, and two to as IIBd and IIBm. Fiber

mATPase

idative

one most

use of both of practically

classes: referred

intensity,

microphotometric

com-

fibers

stained according to the BK method and the GS method. to avoid, as far as possible, the psychophysical difficulties with

show-

KERNELL

ofwhite

light

(6).

Within

each

fiber,

a central

measurement

field

of 12.5

tm diameter was chosen. For the SDH assessment, this meant that the measurements did not include the irregular accumulations of subsarcolemmal staining(i.e., subsarcolemmal mitochondria)(6,13,19,22). Previous studies of fibers from individual rat motor units have shown that there is a good correlation between the present type of central SDH measurement (core SDH)

and

the

sensitivity

to fatigue

(13).

During

our

transmittance

mea-

surements, the field diaphragm was maximally closed. For each fiber, the optical density (D; given as %) of core staining was calculated according to the

equation D

where

the background

mitted side

through

the

muscle

=

100

*

logio

(lo/Ix)

value L. was the measured

glass slide plus mounting tissue,

the center

of the

surements

ofoptical

and

respective

density

Downloaded from jhc.sagepub.com at SWINBURNE UNIV on September 2, 2014

I was the muscle

intensity fiber.

were primarily

intensity

medium oflight

It should

of light

plus coverslip transmitted be noted

introduced

that

trans-

just outthrough our

mea-

as a way of avoiding

MYOFIBRILLAR

many

ofthe

ATPase:

FOUR

FIBER

psychophysicalproblems

of relative

staining

TYPES

associated

591

with

subjective

judgements

Ac-ATPase

intensity.

For individual

General

Comment

on Classification

in a scatter

Procedure

isons

Terminology

A major

in the present

staining

analysis

according

concerns

to two different

the classification

starting point. Hence, our approach tially classify fibers into Types I, hA,

will and

ATPase

analyze

reactions,

of fixed

and (b) secondarily

alk-ATPase

(10,11,23)

tend the classification nique. The detailed brachii

techniques,

which

and

muscles

can

essentially be to (a) IIB according to the whether

be used

tibialis

anterior.

will be briefly

and/or

ex-

to the ac-ATPase techon results from bi-

Confirmatory

commented

ac-

the method

to confirm

performed according account will concentrate

mi-

observations

from

on at the end ofthe

Results

section.

When

According

prepared

by the

almost (Figure

to the

BK method

alkaline (pH about about 4.5 or less),

and

are light II fibers

(Figures

that

fibers

after alkaline the opposite

a clearly ic, 2A,

have

portions

9.4 or more) or acid fibers of mixed limb

treatment and dark after acid, and for Type is true (1). This was also the case for the pres-

pH

10.4),

of Type

The

see Methods)

intermediate

fibers

ofType

hA

la and

2A)

(Figures

two muscles

we chose

tenor,

biceps

brachii)

spect

to the

topographic

(Figures

technique,

la and

were

markedly

distribution

ib) (for tibialis

IIB, and lightly

stained

analysis

(tibialis

heterogeneous

within

different

fiber

re-

types

see 13, 21). In the follow-

ing, we will define as “red” the portion containing (Figure la) and as “white” the portion lacking such

Type fibers

One white and one red sample were analyzed for each muscle. white samples were taken from regions relatively remote from

the

red portion,

, the white

samples

were

just

the

portion.

i.e. zone

outside

red

not

located

According

In accordance GS method

with

within

(Fixed

three

distinct

(Figures

descriptions

alk-ATPase intensities ic and 2C).

technique; ofmATPase

(11,23),

a marked IIBm

staining

method

indeed

regions,

IIBm fibers

in the same

in ac-ATPase

delivered,

were

clustered Within

as noted

the GS method(fixed of group intensity

above, la, two

alk-ATPase)

IIB into a dark and an in(Figures ic, id, 3A, and

we will refer

(moderate). belonged

heterogeneity

fibers;

to these

new subgroups

In our fixed alk-ATPase prepato the same optical density clus-

four

techniques

for mATP-

main

Figures

between

clusters

1 and

all the theoretically number of fibers

by the GS method

and

the BK and GS methods,

of staining

3) represented

patterns only

possible combinations with a clearly “aberrant”

was very

(IIBd

4b). comparisons

observed

IIBm;

was revealed

Figure

In the present the

small

(0.3%;

(I,

hA,

a limited

IIBd,

fraction

of

ofstaining intensity. The or “unusual” staining

l#{224}ble1). Thus,

graphs

of the

kind

shown in Figures 3A and 3B demonstrated that the relationship between the staining patterns produced by the BK and GS methods highly

structured;

there

of random

lack

were

IIBd red

fraction than

ofthe

within

IIB fibers white

no

indications

for

of correspondence

Both of the new subtypes within all the analyzed muscles

Relationship

(see

was relatively

more

portions

any

important

Introduction).

of IIB fibers were (l#{224}ble1). Within

muscle

of the

Between

Oxidative

made

see Materials

fibers

distribution of staining intensities (Figures When considered together, however, the

purposes,

(dark)and the IIBd

Enzyme of muscle

significantly

activity our version

classified

darkest

compar-

BK method

ones in fixed alk-ATPase, and vice versa exceptions, see Table 1). However, fibers

red as well as white

Groups

to the GS Method

earlier

direct

I by the

ase was particularly evident to the eye when scrutinizing white musdc regions: large areas within such muscle portions might look completely homogeneous to the BK method (all IIB; Figure 4a)whereas

and

Staining

Such

as Type

we techother

well represented each muscle, the dominant

(Figure

within

1; Table

1).

I fibers (Figure

lb). The

a transitional

3B) (27).

consistently

the

caused an evident subdivision termediate category of staining

was

an-

with

sections,

revealed the presence of four distinctly different categoin red muscle portions. This was due to the fact that,

degree

of the

anterior

pro-

(1).

for our detailed both

that

intensity: dark fibers of pH 4.5 or the light ones

serial

of fibers

ries

pattern

our ac-ATPase

distinct clusters of staining fibers as the dark ones after

been (9):

each

trimodal and 2C).

methods

ally around

4.7;

3A and

categorized

GS method

red muscle

and

duced three Type I (same

through

ofthe two mATPase staining optical densities against each

classified as Types IIA and IIB by the BK method were in a different manner when stained by the GS method.

ent muscles, i.e., we could produce an unequivocal classification of Type I vs II fibers. Furthermore, by careful adjustment of the acidity ofpre-incubation we could in all casesfind a pH value (gener-

portions

followed

ter as the IIA fibers (Figure 3A). The difference between the two staining

pre-incubated

muscles are known to show only two distinctly different staining intensities. Furthermore, the alkaline pre-treatment produces an inverted version of the strongly acid pre-incubation: Type I fibers

fibers

were

always the lightest 3A; for occasional

as IIBd rations,

to the BK Method

according

at a pH that is markedly (in our preparations pH

caused

diagram

3B). For descriptive

Staining

after

that

also generally

way

are associated with two different fiber type terminologies (see Introduction). To simplify the description we will, in the following, use the methods and terminology of Brooke and Kaiser (1) as a

other

fibers

showed

would

question

ofmATPase

ceps

Alk-ATPase

Fixed

compared the staining reactions niques by plotting the respective

Results

and

vs.

of SDH the

new

Staining

and

Activity

fibers

of Types

different

from

(Figures and

mATPase

le,

IIBd each

if, 3C,

unexpected

and other

3D,

observation

the IIB subtypes,

the average

and Methods)

for fibers

located

within

in red muscle

the white the IIBm

portion (Figure 5). This tendency fibers than for those categorized

Downloaded from jhc.sagepub.com at SWINBURNE UNIV on September 2, 2014

and

SDH

staining

the red muscle

IIBm

were

with

respect

distinctly to their

5). Furthermore, that,

for each

was significantly

portion

than

for those

we one

of

darker from

was more marked for as IIBd, i.e. , the ratio

592

LIND,

y

KERNELL

.

.-. ::.c-..’ :

.

,

%..-

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- -

;j±i

.%

‘ ..

‘J1

...

.

:

‘

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.

I.

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:-

,-.. .1

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r ,:

:.i:,

1,;.

:-

..-

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.

;. .

MYOFIBRILLAR

ATPase:

FOUR

FIBER

TYPES

593

Figure 2. Histograms showing the distribution ofoptical density of mATPase staining among 100 fibers measured from each one of the two muscle portions represented in Figure 1. (A,B) Staining for ac-ATPase (of. Figures la and ib). (C,D) Staining for fixed alk-ATPase (of. Figures ic and ld). A and

‘White’

B

region

50 12

.

4) .0

4)

“.4

,-

0

0

z

C are from red muscle portion; B and 0 are

z

25

from whfte muscle portion. Both graphs from a given muscle portion show data for the same individualfibers(serial sections). Units ofoptical densitycalculated according to the equation in Materials and Methods.

0 0

25

50

Ac-ATPase

Ac-ATPase

C

D

40

50

12

12 4)

.0

.0

0

0

z

z 20

25

0

0

0

40

80

Fixed

between

the

SHD

staining

of IIBd

and

IIBm

fibers

was,

on aver-

age, significantly greater within white than within red muscle tions (Figure 5). For all the muscles in Figure 5 and l#{224}ble1, the average

SDH

staining

(Fig-

was consistently

ure 3C); statistically

ranked

such

all these type-related significant (t-test,

that

IIA>I>

por-

IIBd>IIBm

differences in SDH p