0022-

1554/79/2710-

THE

JOURNAL

Copyright

1357$02.00/O OF

©

HISTOCHEMISTRY

by The

1979

AND

Histochemical

Horseradish

Vol.

CYTOCHEMISTRY

Society,

WEIHE,

Department

pp.

10,

as a Tracer

HARTMUT

NIMMRICH,

ofAnatomy,

1357-1359,

1979

Printed

Peroxidase

EBERHARD

No.

27,

Inc.

University

for

JURGEN

ofHeidelberg,

Capifiary

METZ Im

Permeability

WOLF

AND

Neuenheimer

GEORG

Feld

307,

Studies

FORSSMANN

D.69

in IL S.A.

(BR

Heidelberg

79-166)

(Germany)

The permeability of capillaries was investigated utilizing an in vivo injection of horseradish peroxidase (HRP) and an in situ perfusion ofa balanced salt solution containing HRP and lanthanum chloride. In the continuous capillaries of heart and muscle, HRP diffuses mainly through intercellular junctions, while in testicular capillaries, the transport is via micropinocytotic vesicles. The diffusion and micropinocytotic transport of HRP was demonstrated in both directions, i.e. from the capillary lumen to the interstitium and vice versa. Lanthanum can be used as a bidirectional inhibitor of micropinocytosis. The transport of HRP is then almost completely hindered in testicular capillaries. In heart muscle, the effect on HRP transport is not significant, due to second transport pathway, i.e. intercellular cleft passage.

Three the

main

techniques

exchange

of

interstitium. in

These

vivo

studies

electron

opaque enzymatic have

(1)

site

capillaries (2,

7,

and 8,

(2)

1 1-13,

specific results

both

of the

passage

the

of the

this

method

present

with

diffusion

and

of tracers

transport

through

MATERIALS

the

AND

pathways capillary

and

capillary

the

One

hundred

well

as

experiments

passage

anesthetized

system

in a dose

carried

out: injected

was

before

jugular

perfusion this

contrast

at

technique,

to the 5 sec

10,

15,

endothelia.

included the perfusion

tions fused

varying

containing

combination The using

reactions Karnovsky

5 sec.

exact

intervals

after

tracer

the

perfusion

the

injected

possible HRP

method.

injection

of HRP

into

injection was

begun.

of HRP and

in

an

and/or

time

tissue

fixation tissue

the

after

method

were ((6),

both

preparation of Forssmann

performed see

also

types for

according

et

al.

as well

microscopy

was

(5). All histochemical

to a modification

structures

15-45

sec.

is

In the

intra-

observed.

3)

45 to 60 sec

homogeneously

distributed

caveolae,

the

early

of filled

after

abluminal

The

lasting

at

in the

intracytoplasmic

interstitial

the (Figs.

passage

space

muscle,

(Figs.

4 and

HRP

is observed.

micropinocytotic

through

was

However, The

passage

vesicles.

inhibition

delayed

capillary

in the

endothe-

capillaries

of transendothelial

the

washout

utilizing

compared

testicular

experiment

Furthermore,

chloride

the

same

significant

interstitium

lanthanum

is observed. its length.

solution is perfused first and followed by a lanthanum it is found that lanthanum

3). In the

no

result

throughout

through

of HRP

1 and

a similar

stained

exclusively

heart ular

in

of HRP

a perfusion

to the

from

the

solution

controls

of the passage

(Fig.

of

testic-

containing

2).

DISCUSSION Traditionally,

1, 2,

second

perfu-

of Graham

and

(4), Majno

and Joris

per-

types.

or

such

in

capillaries

fenestrated subdivisions

solu-

were

sequence

of experiments

electron

is not

points

2) The

LaCl.

alternating

of

of the

(2, 3).

achieved

perfusion, i.e. in situ organ organs. Balanced electrolyte

amounts

the the

(5, 15, 30, 45 sec;

was

hum

II)

to start

At varying

fixation

whole animal of isolated intervals

then

immobilization

fixation

the

time

was it was

Type

cleft

to occur

inhibits

of the

a hydrostatic

(Sigma,

other

number

caveolae

and

of the testis,

intercellular

If a LaCl.4 containing HRP solution containing

of

transen-

aorta to

HRP

Thus,

types

of the

connected

weight

150-260

Two

The as

as the

contain

carried

HRP and

(3)).

continuous

are

necessary

variability

been

of other

classified

to Bennett

our analysis, respect

discontinuous,

components

Forssmann

1 ),

it is evident

to the

and

as

et a!. (

that

intercellular of the

further

junctional

endothelial

micropinocytotic

lining

vesicles

must

(10). show

functionally may

only

results

with

tracer (8)

with channels,

vesicles Karnovsky

have according

(9). From

fenestrations,

also be considered Our results

(14).

subsequent out

g body

different

studies abdominal

injection.

within

weighing used.

The and

tracer

sexes

g were

and cleft,

In the capillaries the

course

HRP:

after

mm)

experiment sion and for

the

immersion

45

25

1 ) Time

a faster

to 45 mm 30,

of both

cannulated

mg/100 vein

fixation

Using

rats

abluminal

ad.luminal

and

5).

METhODS

about

of iv.

of 20-40

internal

Wistar

weighing

animals

perfusion

from

fifty

mice

were

dothelial

5,

and 20

from in the

to be equilibrated

interendothelial

No

of

1) Filling

(caveolae)

15 sec.

ranging

is seen

sequence

observed.

2) Filling of the interstitium, vesicles, as well as the myocar-

increase

HRP

following

was 1 to

phase

and

seems

lumen,

vesicles,

seems g as

vesicles of HRP

the vesicles

within

a gradual

45 mm whereby

least

The

occurs

in the

phase

cytoplasmic

HRP,

this time phase. intraendothelial

occurs

of this

of

interstititum

micropinocytotic

cleft

diffusion

to the

used.

injection

myocardial

open

T-system

stage

vascular

were

vito the

adluminal,

dial

kinetics

different

studies

in

into

interendothelial

certain

be due

an

are labeled during abluminal caveolae,

respect

passage

study,

tracer

the

sized

of

may

of

inves-

with

endothelium

though,

use

studies.

Several

the

After diffusion

commonly

of medium

using

the

the

most

of peroxidase

combined

elucidate

kinetics

In

and

endothelium.

differences,

applied.

procedures

tracers,

RESULTS the

tracers,

the passage

from

demonstration These

and

of radioactive

one

capillary results

concerning

lumen

in ultrastructural

been

passage

the

16).

techniques

perfusion

the

HRP

use

labeled

has

varying

of

the

to demonstrate

through

capillary

tracers

(HRP)

obtained

the

include

enzymatic

tracers

molecules

tigators to

and

our knowledge the

of fluorescent

peroxidase

protein

improved between

techniques

of color

Horseradish

used

have

metabolites

and

1357

Downloaded from jhc.sagepub.com by guest on March 26, 2015

that

the

terminal

open

intercellular

partially

contribute

studies Wissig

are and

in

blood

vessels

clefts.

Thus,

to the

transport

agreement

Williams

(16).

with On

of

the

heart

micropinocytotic

the

of HRP. the

Our

findings

contrary,

of Simi-

.1.1.

:-

‘

.1

-.

‘1

S

.#w

.-

..:.

, -..-(

...

.-

.

....

;.

..4..-.,.

.

.y, ,...

:_..A. -,,i .S

,.r

‘-

?w

.,,‘..

ILLJ

‘.

%);

_#{163}.‘.,.-

5:;t. -

‘

-

4.

. ‘.5 . a,’.,.

S

-

#{149}

‘ .‘.i. . .

-.... .

.;c.

.

.

. . . ... .

.-

--

.

..c.-’ S

-.. 5..

FIG.

I

.

Testis

capillary

5 mm after

perfusion

)

.1

with a solution

containing

I mM lanthanum

seen

chloride

and horseradish

peroxidase

(HRP).

It is

that the tracer penetrates into adluminal micropinocytotic vesicles (caveolae, see arrows) (x15,200). FIG. 2. Testis capillary in the same experiment as seen in Figure 1 at higher magnification. The interendothelial cleft is filled with tracer UI) to the tight junction (arrows) (x35,000). FIG. 3. Testis capillary 5 mm after in vito injection of HRP and subsequent in situ perfusion with a lanthanum chloride containing solution. It is observed that the peroxidase cannot be washed out from the interstitium and lanthanum only diffuses into the outer part of the intercellular cleft (arrows) (x132,000). FI;s. 4 and 5. Muscle capillaries (Fig. 4, heart; Fig. 5, diaphragm) 5 mm after in vivo injection of HRP and short rinsing before perfusion fixation. Note that the tracer is homogenously distributed in the interstitium and the intercellular cleft (arrows) (x 126,000). 1358

Downloaded from jhc.sagepub.com by guest on March 26, 2015

CAPILLARY et al.

onescu

(1 1, 12) did

interendothelial ity

was

inhibited

reduced

by

in testicular

should

be

noted

myocardium,

not

observe

a passage

In experiments

clefts.

lanthanum,

that

through

such

capillaries

were

conduction

system

found

capillary

types,

the

transport

heart

and

both

HRP

muscle

types,

though,

the

is negligible

due

functionally

open,

The

demonstrated studies

and

2)

and

diffusion

the

are

Bennett HS, of vertebrate

2.

Forssmann

combining

Histochemie

8:50a, 1969 A method

perfusion

fixation

20:277,

11.

for

CITED

13.

chez

in

with

vivo

diffusion

le rat et La

intravenous

tracer

tracer

WG:

Die

normale

mene. Med 5. Forssmann

Welt WG,

27:1606, 1976 Ito 5, Weihe

ultrastructural Cytochem

und

Aoki

A, Dym

binding

injection.

15. Weihe

Transportph#{228}noM,

Fawcett

testis.

Anat

Rec

The early stages in the proximal

cytochemistry 14: 291, 1966

of absorption of tubules of mouse

by a new

technique.

J His-

G, Joris

DW:

I: Endothelium

1977:

a review,

The

Thrombotic

1978

Simionescu N, Simionescu M, Palade GE: Structural basis of permeability in sequential segments of the microvasculature of the diaphragm. II. Pathways followed by microperoxidase across the endothelium. Microvascular Research 15:17-36, 1978 Weihe E, Aoki A, Aum#{252}llerG, Greenberg J, Metz J, Hartschuh

studies

as

sites.

Wissig

a

diffusion

Cell

Tim

E, Kalmbach system

192:77-87,

16. E,

for the

Process in Atherogenesis. Edited by AB Chandler et al. Plenum Press, New York, 1978 Simionescu M, Simionescu N, Palade GE: Segmental differentiations of cell junctions in the vascular endothelium. The Microvasculature. J Cell Biol 67:863, 1975 Simionescu N, Simionescu M, Palade GE: Open junctions in the endothelium of the postcapillary venules of the diaphragm. J Cell

duction

Gefdl3wand

method

W, Forssmann WG: Experimentelle Untersuchungen zur terminalen Strombahn des Hodens. Verh Anat Ges 72:577-560, 1978 14. Weihe E, Hartschuh W, Metz J, BrUhl U: The use of ionic

1969

4. Forssmann

kidney:

lanthanum

for

fixation

RC, Karnovsky MJ: horseradish peroxidase

Biol 79:27-44, 12.

of transport

JC: Morphological classification Am J Physiol 196:381, 1959.

La perm#{233}abilit#{233} des capillaires

sours. J Microsc 3. Forssmann WG:

pathway.

pathways,

kinetics

10.

to demonstrate.

Luft JH, Hampton blood capillaries.

WG:

the

capmicro-

of HRP

perfusion

1359

1977

tochem

9. Majno

cleft.

transport

HRP

7. Johansson BR: Permeability of muscle capillaries to interstitially microinjected horseradish peroxidase. Microvasc Res 16:340, 1978 8. Karnovsky MJ: The ultrastructural basis ofcapillary permeability studied with peroxidase as a tracer. J Cell Biol 35:213, 1967

these

these by

suitability

1) the

injected

In

of a second

the

6. Graham

through

In

exchange

whereby

LITERATURE 1.

In

occurs

intercellular

identify: difficult

brain.

clefts.

presence

shows

of transport,

gradients

the

transport

unrestricted

technique

188:307,

working

capillaries

micropinocytosis.

of blood-tissue

to ultrastructurally direction

and

intercellular

to the

in

of a passive diffusion restricted in certain by

the

and

control

pinocytosis

tracer

testis

systems,

vesicles

That

is, the

in the

is controlled

capillary

micropinocytotic

illary

i.e.

only

WITH

An improved

capillary

fenestrated

are present (5). We conclude from our studies a discrimination through the interendothelial clefts as being capillaries,

STUDIES

where

in the

continuous

the

micropinocytotic activHRP transport was significantly but not in heart muscle capillaries. It

capillaries

whereas

PERMEABILITY

and 178:285-302,

tracer

Res

P: Ultrastructure

of the

heart

in various

as a 1977

marker

of

of capillaries mammals.

Cell

calcium

in the

con-

Tiss

Res

1978

SL,

Williams

microperoxidase.

Downloaded from jhc.sagepub.com by guest on March 26, 2015

MC:

Permeability

J Cell Biol 76:341-356,

of 1978

muscle

capillaries

to