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The effect of nerve section on the incidence and distribution of gap junctions in the odontoblast layer of the cat.

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THE ANATOMICAL RECORD 218:458-465 (1987)
The Effect of Nerve Section on the Incidence and
Distribution of Gap Junctions in the Odontoblast
Layer of the Cat
G.R. HOLLAND
Division of Endodontics, Faculty of Dentistry, University of Alberta, Edmonton, Alberta,
Canada T6G 2N8
ABSTRACT
Gap junctions are numerous in the odontoblast layer of the dental
pulp and may link sensory axons to odontoblasts. If these junctions do link axons
and odontoblasts, they, together with the axons, should disappear after cutting the
pulpal nerves centrally. Under general anesthesia the inferior alveolar nerve on one
side of two young adult cats was sectioned. Under general anesthesia the animals
were perfused with fixative 56 hours later and the corona1 dental pulp prepared for
electron microscopy. Ultrathin sections were examined from the level of the pulpal
cornu and levels approximately one, two, and three mm below this. The incidence of
cell processes and gap junctions was measured at different distances from the pulp
predentin junction, and operated and control sides compared.
The odontoblast layer at the level of the cornu differed from elsewhere in having,
on the control side, a greater density of cell processes and gap junctions and in
having clearly recognizable axons approaching to within 5 to 10 pm of the predentin.
The only statistically significant changes after nerve section occurred in this layer
and consisted of a decline in the incidence of cell processes and of gap junctions that
link one cell process to another. There was no significant difference between the
operated and control sides in the number of gap junctions linking cell processes to
recognizable cell bodies.
The odontoblast layer in the pulpal cornu contained substantial numbers of unsheathed axons, many presumably en route to the dentin. These axons may participate in gap junctions that link them to other cell processes, possibly even other
axons. No clear evidence was found of junctions involving axons and identifiable
odontoblast cell bodies.
A synapselike contact between odontoblasts and
nerves would add structural support to two ideas; that
the odontoblast may act as a sensory receptor and that
sensory nerves have a trophic effect on odontoblastic
activity. Other evidence for both these ideas is inconclusive in part due to the technical difficulties in examining
such a remote and well-concealed cell, and in part due
to conceptual difficulties in defining what constitutes a
receptor and what is a direct neurotrophic effect (for
review, see Holland, 1986a).
Electrophysiological methods of recording the activity
of odontoblasts, although promising direct evidence of
excitability and synaptic transmission, are difficult. The
most successful attempt thus far (Magloire et al., 1979)
did not positively establish the receptive role or the
neural link. Neural resection experiments (Avery et al.,
1974)designed to establish a trophic function are fraught
by secondary effects such as the loss of propioceptive
input. Other structural studies have been equivocal.
Conventional anatomical methods are cursed with the
difficulty of identifying unsheathed axons in the processabundant odontoblast layer. The potentially most informative approaches using anterograde transport of ra@ 1987 ALAN R. LISS, INC.
dioactive labels in nerve fibers (Byers and Kish, 1976;
Byers, 1977, 1979) and tracing of nerve fibers in serial
sections (Holland, 80a) could not demonstrate any convincing specialized junctions involving axons. Although
earlier studies (Frank, 1968; Arwill, 1968) had reported
synapselike structures in dentinal tubules, subsequent
observations of labeled axons in dentin (Byers, 1977) or
as a result of comparing intracellular filament and tubule populations (Holland, 1986)could not confirm these
earlier findings.
Certainly anything like a classical chemical synapse
is absent from the odontoblast layer, and in our view
the dentin a s well (Holland, 1986). The only potentially
synaptic junction available in profusion is the gap junction in the odontoblast layer. The gap junction is known,
in the central nervous system, to be capable of acting
synaptically (Pappas and Bennet, 1966). Whereas these
junctions are widely distributed in the peripheral pulp
(Holland, 19771, no study has thus far established with
Received December, 1986; accepted March 24, 1987.
NERVE SECTION AND ODONTOBLAST GAP JUNCTIONS
any degree of certainty that they link axons and odontoblasts. The most positive finding in this direction is
the cytological similarity between the processes linked
to odontoblasts and axons (Holland, 1980b).One obvious
approach, which may establish whether these junctions
involve axons or not, is to examine the effects of nerve
section. Recent studies have examined the effect of denervation on the apical (Holland and Robinson, 1984)
and dentinal (Holland et al., 1986) innervation. This
study examines the changes brought about by shortterm denervation on the distribution of gap junctions in
the odontoblast layer from some of the animals prepared
for these earlier studies.
MATERIALS AND METHODS
Two young adult cats were used in these experiments.
The animals were anesthetized with a mixture of alphaxalone and alphadolone acetate (Saffan, Glaxo Laboratories, U.K.). Body temperature was maintained with
a n electric blanket controlled from a rectal thermistor.
The left inferior alveolar nerve was exposed in the mandibular canal in the region of the anterior border of the
masseter muscle. The nerve was sectioned and the cut
ends replaced in apposition. Each animal was given
penicillin postoperatively. Electrophysiological observations were made on these animals and have been reported elsewhere (Holland et al., 1986). Fifty-six hours
after nerve section, the animals were, under general
anesthesia, perfused with a fixative mixture via the
common carotid arteries. The perfusion was initiated
459
with a prewash of 300 mosmol. pH 7.4 phosphate buffer
(Hayat, 1970) in which 2.7% dextran had been added
(Dextran T40, Pharmacia Fine Chemicals, Sweden: Aldskogius and Arvidsson, 1978). The perfusion was continued and completed with a similar mixture to which 5%
glutaraldehyde had been added. After overnight immersion of the head in buffered glutaraldehyde without
dextran, the lower canine teeth were removed and decalcified in a mixture of 4% EDTA and l%glutaraldehyde. The crowns of the teeth were slit transversely at
1 mm intervals and the resulting discs processed by
washing in phosphate buffer, postfixation in 2% phosphate-buffered osmium tetroxide, en bloc staining with
uranyl acetate, alcoholic dehydration, and embedment
in Araldite resin.
Ultrathin sections of each block from the corona1 half
of the crown were cut. To ensure that sections close to
the pulpal cornu were examined in each tooth, the block
containing the cornu was step sectioned until the pulp
was reached. Ultrathin sections were taken from the
block containing the cornu and the three, 1-mm blocks
below it. A single section containing a complete cross
section of the pulp was cut a t each level from both
control and operated teeth, a total of 16 sections. This
enabled the position of each section to be related to the
pulpal cornu. The sections were stained with uranyl
acetate and lead citrate and examined in the electron
microscope. Montage electron micrographs were taken
of segments of the odontoblast layer from its junction
with the predentin to a level 40 pm toward the central
Fig. 1 , Low power micrograph of a typical area examined. This section was taken 1-2 mm below the
pulpal cornua from a cat whose inferior alveolar nerve on the same side had been transected 2 days
earlier. PD = predentin, OD = odontoblast.
G.R. HOLLAND
460
TABLE 1. Total no. of Drocesses
Distance
from PDJ (pm)
Level
1 Control
operated
2 Control
operated
3 Control
operated
4 Control
operated
5-10
0-5
10-15
68.3 (19.1) 114.0 (21.0)* 114.0 (8.5)*
38.5 (18.8)
35.5 (30.9)
48.0 (11.9)
60.0
(26.2)
46.5 (17.7)" 66.8 (5.7)
50.6 (8.9)
40.6 (20.7)
7.2 (8.5)
56.3 (15.6)
56.3 (10.1)
52.3 (28.5)
51.0 (22.1)
47.0 (6.3)
13.2 (9.4)
31.9 (15.1)
36.8 (16.4)* 54.5 (16.8)
42.5 (14.5)
45.0 (9.3)
9.0 (6.7)
15-20
20-25
25-30
30-35
35-40
89.3 (23.7)*
32.5 (5.0)
54.8 (15.8)
41.6 (11.7)
37.8 (13.1)
27.7 (10.7)
28.8 (13.1)
24.3 (11.4)
82.0 (18.7)"
18.5 (1.0)
44.5 (9.2)
42.2 (18.7)
22.5 (18.1)
29.0 (11.0)
16.5 (3.9)*
28.0 (4.8)
88.0 (9.0)*
15.0 (10.2)
21.5 (4.9)
36.8 (11.5)
19.3 (1.5)
23.8 (9.1)
20.0 (8.5)
30.8 (6.1)
53.7 (47.0)*
22.8 (7.8)
22.0 (8.5)
30.2 (8.4)
14.0 (4.5)
16.3 (2.5)
11.5 (12.0)
15.7 (2.3)
42.0 (21.5)
15.8 (10.3)
23.5 (2.1)
19.2 (4.8)
16.7 (0.6)
12.3 (5.0)
5.0 (0.0)
16.5 (0.71)
*Statistically significantly different (P < 0.05).
140
r
120
t
140
,-
T
I
\
E
2
40
i
-1
0
0
1
2
3
4
Level Below Pulpal Cornu. ( m m )
0
0
I
I
I
I
10
20
30
40
Distance From Pulp-Dentin
Junction ( p m )
Fig. 2. The incidence of cell processes 5-10 pm from the pulp-dentin
junction at different levels in the corona1 pulp. Filled circles represent
control values, open circle counts after nerve section. Bar lines show
standard deviations. Only a t the level 0 to 1 mm below the cornu are
the differences statistically significantly different (P < 0.05).
Fig. 3. The incidence of cell processes at different distances from the
pulp-predentin border at the level of the pulpal cornu. Solid circlescontrol, open-operated. All but the first and last pairs of points are
statistically significantly different (P < 0.05).
pulp. The original magnification was 1 , 2 5 0 ~and with
enlargement a t printing of 4.5 x gave a final total magnification of 5,625 X.
Each montage was subdivided into four strips 20 pm
wide and 40 pm long with the long axis a t right angles
to the pulp-predentin border. Each strip was then subdivided every 5 pm along its length. This resulted in a
series of eight zones at different distances from the predentin each 100 pm2 in area. This resulted in 268 zones,
each localized in terms of distance from the predentin
and position in the tooth. This ensured that in comparing observations on operated and control sides, comparisons were being made from similar locations. Previous
studies have shown that both gap junctions and axons
show regional variations in distribution (Holland, 1978,
1980a).
The following observations and counts were made from
each zone:
The number and length of gap junctions linking one
cell process to another.
The number and length of gap junctions linking cell
processes to cell bodies.
The number and length of gap junctions linking one
cell body to another.
The number of cell processes present.
The number of axons present.
Structures that may have been degenerating nerve
terminals and the relationship of these structures to
other cells and processes.
Cell bodies were defined a s profiles that contained
nuclear profiles. Axons were identified by their relationship with Schwann cells. It was realized that a t the
magnifications used, gap junctions could not successfully be differentiated from tight junctions, but our earlier studies have shown that the vast majority of these
junctions are restricted to the cell to cell contacts adjacent to the predentin. Junctional lengths were measured
using a digitizing pad linked to a small computer (Bioquant, R & M Biometrics, Nashville, TN). The observer
was unaware at the time of measurement of the origin,
NERVE SECTION AND ODONTOBLAST GAP JUNCTIONS
1407
tween operated and control counts were not significant
in 21 of 24 cases.
-
Cell to Cell Junctions (Fig. 5)
Junctions with nuclear profiles on each side were rare;
only 16 were observed in total. The majority were in the
region immediately adjacent to the predentin. Whereas
more were present in the control tissue than the operated, differences were not statistically significant.
120
07
%ul
100-
a,
0
0
&
.c
0
L
461
80 -
T
a,
n
E
3
z
Distance From Pulp-Dentin
Junction ( p m )
Fig. 4. The incidence of cell processes at different distances from the
pulp-predentin border at a level 3-4 mm below the pulpal carnu. There
is no statistically significant difference between control (solid circle)
and operated (open circle) values at any point.
operated or control, or position of the sample under
examination. In all, 2,900 junctions were counted and
measured. In comparing incidence and lengths between
operated and control sides, a simple nonparametric
ranking test, the median test, was used.
RESULTS
No gross differences in appearance were obvious between tissue taken from various levels or between operated and control sides. The conditions of fixation resulted
in a sizeable extracellular space and clear delineation of
cell processes (Fig. 1).No plausible degenerating nerve
terminals were found.
Distribution of Processes (Table 1)
At different levels in the crown (Fig. 2)
In control teeth the largest concentration of processes
was at the level of the pulpal cornu. The incidence declined in the more apical levels. In operated teeth there
was no consistent pattern of change between different
levels; cornual counts were not significantly higher than
those at lower levels.
At different distances from the pulp-predentin border
(Figs. 3,4)
In control teeth there was a decline in the number of
processes from the pulp-predentin junction toward the
central pulp. This was particularly marked a t the highest level near the cornu (Fig. 3). At lower levels (Fig. 41,
the decline was much less marked. The pattern in operated teeth was similar, but at the cornual level the
number of processes was always lower than in the control teeth and the difference was statistically significant
(Table 1).At the more apical levels, the differences be-
Process to Cell Junctions (Fig. 6)
The number of junctions linking processes to clearly
recognizable cell bodies in control tissue declined in the
coronal to apical direction, although most of the decline
occurred between the level of the cornu and the next
level apically. There was no similar regional pattern in
teeth from the operated side. At the level of the cornu,
peripheral regions adjacent to the predentin showed
higher incidences than did more central areas (Fig. 7),
although this pattern was not evident a t lower levels
(Fig. 8). The incidence of these junctions in the cornual
level was always lower in operated than in control sides;
however, a t only one point were these differences statistically significant (Fig. 7). There was no consistent pattern of difference between operated and control sides at
lower levels (Fig. 8).
When total lengths of cell process junctions were compared, a similar pattern was found.
Process to Process Junctions (Table 2; Fig. 9)
In unoperated teeth the incidence was highest in the
level immediately adjacent to the cornu and declined
apically, with most of the decline occurring within the
first millimeter. The incidence also declined from peripheral to central pulp (Figs. 101, but again the fall was
a step rather than a slope.
After denervation the incidence of process to process
junctions fell, but only in the most coronal level where
in the region 10 to 35 pm from the pulp-predentin junction differences between counts on operated and control
sides were statistically significant (Fig. 10).
When the distribution of process to process junctions
was expressed as their total length per 100 p area, the
pattern was similar. Significant differences between operated and control sides were again limited to the juxtacoronal level.
Axons
No axons were recognizable in the first 5-pm strip
adjacent to the predentin a t all levels. They first appeared 5-10 pm from the predentin a t the most coronal
level. A general increase in number occurred toward the
central pulp, with the highest counts in the most central
and apical levels. Axons were present in operated teeth,
but none were closer to the predentin than 20-25 pm.
Counts in operated teeth were lower than in control, but
low numbers precluded valid statistical comparison.
DISCUSSION AND CONCLUSIONS
The data from unoperated teeth described quantitatively some of the regional patterns within the odontoblast layer and peripheral pulp at different levels in the
tooth. It is clear that the layer in the cornu was substantially different from the layer elsewhere. The incidence
of cell processes is higher, as are the numbers of junc-
462
G.R. HOLLAND
Fig. 5. Two odontoblast cell bodies (OD) linked by a gap junction (G) and also by a desmosomelike
junction (D) (operated side).
Fig. 6. A cell process (CP contacting a n odontoblast cell body (OD) by a gap junction (arrow) (control
side).
NERVE SECTION AND ODONTOBLAST GAP JUNCTIONS
463
14
12
10
8
4
6
T
4
T
2
0
101
20’
- - 30
0
0
40
J.
Distance From Pulp-Dentin
Junction (pm)
Distance From Pulp-Dentin
Junction ( p m )
Fig. 7.The incidence of gap junctions linking processes to nuclei
containing cell bodies at the level of pulpal cornu,
only one
15-20 ~m from the predentin are control (open circles) and operated
(closed circles) statistically significantly different (P < 0.05).
Fig. 8. The incidence of gap junctions linking cell processes to cell
bodies containing nuclei 3 to 4 mm below the level of the pulpal cornu.
There are no statistically significant differences between control (open
circle) and operated (closed circles) sides.
TABLE 2. No. of process to process junctions
Distance
from PDJ (pm)
Level
1 Control
operated
2 Control
operated
3 Control
operated
4 Control
operated
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
11.0 (10.1)
5.5 (7.7)
15.0 (7.3)*
1.8 (3.0)
13.8 (14.8)
0.7 (1.6)
6.7 (8.1)
1.8 (1.7)
28.3 (13.4)
14.3 (11.4)
14.8 (7.0)
11.4 (10.1)
11.3 (8.8)
9.8 (5.5)
10.0 (11.0)
4.8 (1.5)
25.3 (2.5)*
4.5 (3.1)
17.0 (7.8)
12.0 (7.3)
9.5 (7.0)
12.8 (6.0)
6.8 (3.7)
5.8 (3.1)
27.0 (2.6)”
6.8 (2.2)
7.0 (5.0)
10.6 (8.2)
7.8 (4.9)
12.8 (3.9)
8.7 (7.0)
2.8 (2.5)
14.0 (O)*
5.3 (3.0)
9.3 (5.3)
12.2 (6.6)
2.0 (O)*
11.2 (8.7)
3.0 (1.2)
4.5 (3.4)
15.5 (3.2)*
6.8 (5.7)
4.0 (2.9)
11.8 (6.3)
2.5 (2.5)
8.0 (6.3)
6.3 (4.0)
9.0 (5.4)
15.3 (12.9)*
7.0 (5.5)
3.0 (1.4)
6.4 (5.2)
2.5 (1.9)
6.3 (5.2)
3.0 (4.2)
1.7 (1.5)
17.3 (21.6)
4.0 (3.2)
8.0 (2.9)
5.4 (3.4)
3.3 (4.2)
2.0 (2.3)
2.0 (2.8)
1.0 (1.4)
*Statistically significantly different (P < 0.05).
tions linking these processes to cell bodies or other processes. Although there were no morphologically recognizable axons in the first 5-pm strip adjacent to the
predentin a t any level, they extend most nearly to the
dentin at this level. We have previously demonstrated
that the juxtacornual dentin is the most densely innervated (Holland, 1981; Holland et al, 1986) and it seems
reasonable to suggest that a t least some of the additional processes counted are unsheathed axons.
The effect of nerve section is also only measurable in
the cornu. The reduction in the number of cell processes
after denervation is statistically significant, indicating
a loss of axons. Whereas not all apical axons have degenerated by 56 hours postsection (Holland and Robinson,
1984), a great many do, and as there is a virtually total
loss of intradentinal axons at this time (Holland et al,
1986), it would appear that degeneration occurs not only
centrifugally from the site of section but centripetally
from the terminals. The reason for selecting the short
survival period was twofold. First, to try and catch degenerating axonal figures. This failed. The second reason was to minimize the secondary effects of denervation
that may result from changes in blood flow and loss of
sensory input. We have no assurance, however, that any
of these effects (e.g., increased synthetic activity of the
odontoblasts) did not occur and that the morphological
changes measured were not related.
Whereas nerve section causes a clear effect on the
incidence of cell processes, the effect on the distribution
of junctions is less obvious. No statistically significant
reduction in the number of process to cell junctions
occurred, suggesting that the processes involved in these
junctions do not belong to the proportion of the population that were lost (i.e., were not axons). The reduction
in cell to cell junctions was significant a t the cornual
level. One interpretation of this would be that these are
G.R. HOLLAND
464
Fig. 9. Cell processes linked by gap junctions (arrows)(operated side).
axon to axon junctions. Matthews has presented neurophysiological evidence of coupling (Matthews and Holland, 1976) of axons supplying the dental pulp. This
current data would seem to support the direct gap junction as the anatomical substrate of this phenomenon.
The cornual odontoblast layer is a special region possessing a n abundance of unsheathed axons, some of
which may participate in low resistance gap junctions
with other axons. No statistically defensible evidence
for the innervation of the odontoblast was obtained.
ACKNOWLEDGMENTS
I would like to thank Dr. P.P. Robinson for preparing
the animals, Mrs. Enid Pehowich for her skillful technical assistance, and Mrs. Holly Ridyard for expertly typing the manuscript. This study was supported by the
Alberta Heritage Foundation for Medical Research
(grant #EG-3098) and the Medical Research Council of
Canada (grant #MA-9681).
LITERATURE CITED
I
0
Distance From Pulp-Dentin
Junction ( p m )
Fig. 10. The distribution of junctions linking one cell process to
another at the level of the pulpal cornu. Control values (open circles)
and operated values (solid circles) are statistically significantly different between 10 and 35 Fm from the predentin.
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137:495-508.
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