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Acta Otolaryngol (Stockh)

1993; 13: 98- 101

Ultrastructural Changes

of

the Basement Membrane Zone

in Benign Lesions of the Vocal Folds

F. G. DIKKERS, C. E. HULSTAERT,2

J.

A. OOSTERBAAN2

and F.

J.

CERVERA-PAZ *

From the Depa rtments of Otorhin olaryng ology, and Histo logy and Cell Biology, Un iversity o Groningen,

The N etherlandr

Dikkers

FG,

Hulstaert CE, Oosterbaan

JA,

Cervera-Paz

FJ. Ultrastructural changes o the basement membrane zone in

benign lesions

o

the vocal foldr.

Acta Otolaryngol (Stockh) 1993; 113: 98-101.

The basement mem brane zone (B MZ ) of the epithelium of the vocal folds was investigated electron microscopically in 10

patients suffering from various benign lesions and in 3 controls. Various defects were observed: a thickening by deposition

of

electron dense material, a loss

of

normal architecture, and a near absence of normal hemidesmosomes and anchoring

fibers. Beside these previously reported phenomena, many vesicles carrying electron dense material were found near the

plasma memb rane. The vesicles were observed a t various stages of fusion with the plasma membrane, on the other side

of

which their content was discharged.

In

the cytoplasm

an

increase of mitochondria was seen. The am ount of condensed

chromatin decreased while the nucleoli increased in comparison with the controls. These observations are suggestive of a

hyperactivity of the basal cells

of

the epithelium in response to vibratory stress.

Key words: electron microscopy, vocal cord,

Reinke edema, vocal fold nodules, vibratory injury.

INTRODUCTION

Basement membranes (BM) represent an extracellular

scaffold needed for the orderly development of a

distinct tissue pattern. Among their possible functions

structural support, cell attachment and selective filtra-

tion may be suggested. Some, if not all, of the com-

ponents present in the BM are secreted by the cells

that are attached to it

( l ) ,

and presumably these

components assemble extracellularly

(2).

The basement membrane zone (BMZ) is divided

into four major structural areas (1, 3, 4): the plasma

membrane (PM) of the basal cell of the epithelium,

the lamina lucida (LL) or lamina rara, the lamina

densa (LD), and the sublamina densa (SLD).

Hemidesmosomes (HDs) are special areas where

the basal cell is fastened to the BM. HDs consist of

an attachment plaque, the PM of the basal cell, and

a fibrous network connecting these cellular structures

to the BM. Anchoring filaments (AFs) are specialized

structures which can usually be found in the SLD 5 ,

6).

They connect the attachment plaque to the LD,

and thus hold the basal cell to the BM. The LD is

fastened to the superficial layer of the lamina propria

by anchoring fibrils, probably only by physical en-

trapment (4).

Abnormalities in BM biosynthesis and metabolism

are a sign of several disorders, ranging from

metabolic to neoplastic and from inherited to im-

munological disorders (1,

7).

In order to obtain in-

sight into possible causes of the developement of

Erasmus Programme Fellow from the Faculty of Medicine of

Cadiz, University of Cadiz, Spain

benign lesions of the vocal folds, i.e. cysts, polyps,

Reinke edema or vocal fold nodules, which we have

clinically defined previously

(8),

the BMZ

of

excised

tissue of patients suffering from these lesions was

studied electron microscopically.

MATERIAL AND METHODS

Sixteen pathological vocal folds, belonging to 10 pa-

tients, were investigated. Biopsies from 13 of them

could be used for ultrastructural examination. They

suffered from various benign lesions of the vocal

folds, clinically diagnosed and histologically confi-

rmed. The clinical diagnoses were Reinke edema (4

vocal folds), broadbased thickening (4), vocal fold

nodules 2), polyp

(l),

granuloma (l), and cyst 1).

As controls, biopsies from the vocal folds of

3

males were taken. Two of these were men suffering

from a T3 and a T4 supraglottic carcinoma, respec-

tively, clinically diagnosed and histologically confi-

rmed. Their true vocal folds looked healthy; tissue

had to be taken to confirm the clinical impression

that the vocal folds were indeed free of disease. Both

patients were to undergo total laryngectomy immedi-

ately after ten times 3 Gy radiotherapy. The third

control material was obtained from a boy suffering

from juvenile papillomatosis, an epithelial disease the

bulk of which is made up of a thickened prickle-cell

layer

(9)

leaving the basal layer intact.

Excised tissue was immediately fixed in

2

glu-

taraldehyde in

0.1 M

phosphate buffer, pH 7.4 after

which the tissue sample was split into two parts. One

part was examined light microscopically for histologi-

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Acta tolaryngol Stockh) 113

Basement membrane zone in lesions

of

vocal

folds 99

cal diagnosis, and the other part was prepared for

ultrastructural examination. After glutaraldehyde

fixation the tissue blocks were rinsed in 6.8 saccha-

rose in 0.1 M phosphate buffer, pH

7.4

and postfixed

in 1

OsO

in 0.1 M phosphate buffer, pH 7.4.

Subsequently the samples were dehydrated in an alco-

hol series and embedded in Epon

812.

After staining

in 10 uranyl acetate in methanol, ultrathin sections

were examined in an Akashi 002A electron micro-

scope.

RESULTS

In controls, the shape of the BM was more or less

rectilinear (Fig. 1) whereas in pathological tissue the

BM

had a tortuous appearance (Fig. 2).

The BMZ of pathological specimens showed a

non-uniform thickening which is produced by the

accumulation of an electron dense substance; some-

times

it

seemed as if it was organized in layer-like

structures (Fig. 2). In most of the specimens there

were large areas where the

BM

was thickened (Fig.

3),

but also in some of the areas, the BM had a

normal appearance. The thicker the BMZ was, the

more destruction of the architecture was found.

In almost all of the pathological specimens, vesicles

containing an electron dense material could be ob-

served in a large quantity (Fig. 3); these vesicles were

seldom found in controls. Fusion of these vesicles

with the PM of basal cells and exocytosis of the

electron dense material were frequently observed

(Figs. 3 and

4 .

The HDs in pathological specimens seemed to have

lost their normal architecture in many areas, and they

Fig. 2

Tortuous appearance of BM in pathological tissue.

BMZ showing a non-uniform thickening, produced by the

accumulation of an electron dense substance; organization

in

layer-like

structures.

Bar equals Clm.

Fig. 3

Large areas with thickened BM in pathological

tissue. Large quantity

of

vesicles containing an electron

dense material. Fusion of these vesicles with the PM of

basal cells and exocytosis of the electron dense material

(arrow). Bar equals 1 pm.

showed capricious shapes. Normal HDs were found

in a smaller quantity than in control tissue.

In addition to the findings mentioned above, an

obvious decrease of condensed chromatin in the basal

cells of the pathological specimens was found. The

nucleoli were enlarged and had also increased in

number (Fig.

5) .

Fig. 1

controls. Bar equals

1

pm.

More

or

less rectilinear shape of BM (arrows) In

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100 F.G.

Dikkers

et al. Acta O tolaryngol

Stockh)

113

Fig. 4. Fusion

of

vesicles

with the

PM

of

basal cells and

exocytosis of

an electron dense

material (arrows). Bar

equals 0.1

pm.

Finally, a greater amount of mitochondria could be

seen (Fig. 5 in the patients tissue.

DISCUSSION

Ultrastructural characteristics of tissue from a patient

suffering from Reinke edema have previously been

described, but no mention was made of the changes

in the

BMZ

(10). In another study, polyps and vocal

nodules were examined electron microscopically by

Kotby and co-workers (11, 12). Here the

BM

was

lacking in many parts in the vocal nodules; in other

parts thickening of BM occurred. In the epithelial

cells, variable degenerative signs were observed such

as cytoplasmic vacuoles, attenuated cell junctions and

distorted desmosomal junctions. In polyps, these

changes were apparent only in a few confined areas.

The thickening of the BMZ has also been described

by other authors (13); apparently reduplications of

the LD are responsible for this thickening, as well as

a disorganization of the attachment plaque and dis-

ruption of AFs, which have been related to vibratory

damage of the vocal folds (13). The observations that

AFs are very seldom found (13) could be confirmed

in our study. When visible, they were located in the

areas which appeared more normal.

Frequent occurrence of pinocytotic vesicles along

the

PM

of epidermial basal cells has been described

previously (3). However, no vesicles have been ob-

served in laryngeal epithelium, and none of the stud-

ies cited above mentioned vesicles occurring in cells

of

studied laryngeal benign lesions.

Fig. 5 Decrease of condensed chromatin in nuclei of the

basal cells of pathological specimens. Increase

of

number of

mitochondria. Bar

equals

1 pm.

Although the role of the vesicles we found near the

PM

is not yet clear, we offer the hypothesis that they

are part of a pathophysiological mechanism in re-

sponse to vibratory stress. It might be an aberrant

way of healing, or simply the only way in which basal

cells can respond to the detachment from the superfi-

cial layer of the lamina propria caused by phonatory

injury. According to this hypothesis the exocytosis of

electron dense material contained in the vesicles

would be responsible for the thickening of the BMZ.

Hyperactivity and a higher turnover of the basal

cell layer of the vocal fold epithelium are suggested

by the following observed phenomena: a decrease of

the amount of condensed chromatin, an increase of

nucleoli, an increase of vesicles and an increase of

mitochondria. These findings, together with the BMZ

alterations, might be the stereotypical reaction to

vibratory trauma in all the benign lesions studied.

Further investigations in this field should eventu-

ally confirm our pathophysiological hypothesis of the

changes that phonatory stress induced in the vocal

fold epithelium. Future investigations should help to

determine whether the electron dense material carried

by the vesicles is responsible for the thickening of the

BMZ, and whether this inappropriate response can

be stopped in early lesions.

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Basement membrane

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Manuscript received March 2, 1992; accepted May 8, 1992

Address for correspondence:

Frederik G. Dikkers

Department of Otorhinolaryngology

University Hospital Groningen

Oostersingel 59, 9713

EZ

Groningen

The Netherlands