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The effect of buffer molarity on axonal exposure and axoaxonal apposition in the rat molar pulp.

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T H E ANATOMICAL RECORD 201:471-476 (1981)
The Effect of Buffer Molarity on Axonal Exposure and
Axoaxonal Apposition in the Rat Molar Pulp
G. R. HOLLAND
Departments of Anatomy and Oral Biology, Uniuersity of Manitoba,
Winnipeg, Manitoba, Canada R3E OW3
Axons in the rat molar pulp have been examined morphometriABSTRACT
cally to determine axonal size and the degree of axonal exposure and axoaxonal apposition in tissue fixed by perfusion using 2% glutaraldehyde in cacodylate buffers ranging in molarity from 0.025 M to 0.4 M. Between 31.2% and 45.0% of the
axons were incompletely ensheathed. This proportion of axons exposed was linearly related to the buffer molarity ( P < 0.05) and was approximately double that
found in more central axons. Between 32.3% and 45.0% of the axons were in contact with other axons. This proportion was not linearly related to buffer molarity
but was at least ten times higher than that observed in more centrally positioned
nerve fibers in the inferior alveolar nerves. Increasing buffer molarity reduced the
size of the axons, a relationship not found in the more central axons. It is suggested that axonal exposure and axoaxonal apposition are constant, significant
features of pulpal nerve fibers that may be related to the onset and spread of nociceptive activity. The permeability properties of pulpal axons may differ from
those of more centrally placed axons.
Axons in Raschkow's subodontoblastic plexus show two structural features which may be
related to their role in nociception (Holland,
1980a).One is their incomplete ensheathment
and resultant exposure of the axolemma to the
extracellular space and the other is the close
contact between many of the axons. These features are not unique to the terminal portion of
nerve fibers (Gasser, 1955; Ochoa, 1976). Our
recent study of these arrangements in the inferior alveolar nerve (Holland, 1981) showed
that while up to a third of the axons may be
partially exposed, less than 4% were apposed
to another axon with no intervening tongue of
Schwann cell cytoplasm. The number of axons
exposed was related to the molarity of the solution in which they were fixed, although even in
high molarity solutions which allow the least
exposure 10% or more of the axons remained
incompletely ensheathed. The hypothesis that
axonal exposure and axoaxonal apposition are
particularly extensive in the terminal innervation of the dental pulp has been tested by subjecting the unmyelinated nerve fibers of the
dental pulps from the animals used in our earlier study to a similar quantitative analysis.
MATERIALS AND METHODS
Tissue preparation
Eighteen rats from three litters, all 5-6
weeks old and weighing 200-250 gm were used in random order. Each was anesthetized
with sodium pentobarbital and then perfused
via the ascending aorta for 1 minute with a
prewash of normal saline containing 1%procaine hydrochloride and 0.08 gm of heparin1
liter and then for 15 minutes with a fixative
mixture. In each case the perfusate was maintained at 4OC in a water bath.
The fixative mixture used consisted of 2%
glutaraldehyde, all from the same batch and
manufacturer (TAAB Laboratories, U.K.), dissolved in cacodylate buffer. Three animals
were perfused with fixture mixtures containing each of the following buffer concentrations: 0.025 M, 0.05 M, 0.1 M, 0.2 M, 0.3 M,
and 0.4 M.
The osmolarity of the buffers and the final
Received March 9, 1981; accepted May 7. 1981
G.R. Holland's current address is Department of Endodontics,
College of Dentistry, University of Iowa, Iowa City, Iowa 52242.
0003-276X/81/2013-0471$02.00
0 1981 ALAN R. LISS, INC.
472
G.R. HOLLAND
fixative mixtures were all measured using a
osmomiter (Wescor 5100 B, vapor pressure
type). The values are recorded in Table 1. The
pH of each mixture was measured and ranged
between 7.0 and 7.3.
The mandibular molar teeth were removed
from each animal and left overnight at 4°C in
the same fixative mixture as was used for the
perfusion. The teeth were then decalcified in
EDTA (Warshawsky and Moore, 1967) and
processed according to the following schedule:
Two 10-minute washes in buffer identical to
that used in the perfusion; 2 hours in 2% osmium tetroxide in distilled water; ethanolic dehydration with 2% uranyl acetate in each of
the alcohols; and infiltration and embedment
in Araldite.
Sections in the gold-silver range were cut
from the pulps of the molar crowns and
mounted on uncoated 100-mesh copper grids.
Microscopy
The sections were examined in a Hitachi
HUl2B electron microscope at X 15,000 magnification. Micrographs were taken of at least
100 nonmyelinated nerve fibers from each
group. Each negative was printed at X 2.7
magnification to give a final magnification of
X 40,500.
Measurements
Measurements were made using a "Smart
One Magic System" (Talos Systems, Inc.,
Scottsdale, AZ) which consists of a high-resolution graphics tablet and a microprocessor
programmed such that irregular perimeters
and distances can be measured reproducibly
and accurately by tracing with a cross-hair
sensor. The data may be fed directly into a
larger computer for storage and subsequent
processing. The following parameters were
measured:
The circumference of each axon.
The length of axolemma exposed to the extracellular space.
The length of the axolemma in contact between apposed axons.
For each group of animals the following data
were either derived from the above or did not
require the use of the digitizing system.
The mean axon circumference.
Proportion of the axons that were "exposed."
Proportion of the total axolemma that was
exposed.
The mean circumference of the exposed axons.
BUFFER MOLARITY EFFECT ON AXONS IN RAT MOLAR PULP
473
same animals is also shown on the same axes
(lower line). When the points for the inferior alveolar nerve are compared with those for the
pulpal axons in a paired T-test the difference
is statistically significant ( P< 0.01). The proportion of pulpal axons exposed was always
greater than 30% - the maximum proportion
reached by axons in the inferior alveolar
nerve.
RESULTS
The relationship between buffer molarity
Figures 1-4 show typical nonmyelinated and exposure was even stronger when the denerve fibers fixed in mixtures containing four gree of exposure was expressed as the percendifferent buffer molarities. In all mixtures the tage of the total axolemma exposed ( P <
plasma membranes were intact and the basic 0.01). On average between 6.8% and 15.4% of
organelles readily recognizable. The cyto- the total axolemma was exposed. At all
plasm of some axons fixed in the lower molari- molarities the mean circumference of exposed
ty solutions was less electron dense than other axons was significantly lower than of the total
cytoplasmic matrices, giving the axons a rela- population of axons ( P < 0.05).
Many axons were apposed to one another
tively empty appearance (arrow, Fig. 1). This
did not occur in solutions containing buffer at (between 32.3% and 45%). The proportion of
0.2 M (Fig. 2) or above (Figs. 3, 4). Mitochon- axons apposed was not related linearly to the
dria were often disrupted at very low molarity buffer molarity. The mean circumference of
(0.05 M or less, Fig. 1)but were well preserved the apposed axons was significantly less than
at 0.2 M or above. Microtubules and microfila- the mean circumference of the total populaments were well preserved in all mixtures. The tion of axons ( P < 0.01) and also significantly
gap between axons and the surrounding less than the mean circumference of the exSchwann cell cytoplasm appeared to widen as posed axons ( P < 0.01). There was no signifibuffer molarity increased. The electron densi- cant linear relationship between the size of the
ty of the Schwann cell cytoplasm increased in apposed axons and buffer molarity when
the same direction though no similar effect on tested by regression analysis.
the axoplasm was discernible. The axons in
DISCUSSION
low buffer molarity were commonly circular in
I t is reassuring that reasonable ultrastrucprofile, assuming a more irregular, flattened
tural detail can be preserved over such a wide
outline in the stronger mixtures.
The numerical data for each parameter is range of buffer molarities. Without knowing
summarized in Table 1. Table 2 shows the what the living ultrastructure is it is imposresults of a regression analysis by which the sible to suggest which fixed image most closelinearity of the relationship between each par- ly resembles it.
A high proportion of the pulpal axons are inameter and buffer molarity was tested.
The mean axon size measured as circumfer- completely ensheathed. Although this is deence was always between 1.5 and 2.1 pm pendent on buffer molarity it does not fall
(Table 1).The mean size resulting from pool- below 30% and is always much higher than
ing the measurements for the three animals in that found in the inferior alveolar nerve. We
each group became lower as buffer molarity in- may conclude that axonal exposure is a concreased. The relationship between buffer mo- stant and significant feature of the terminal
larity and mean axon circumference tested by innervation of the pulp and speculate that in
regression analysis was statistically signifi- the absence of any more specialized receptor
(Holland, 1980a) this exposed membrane may
cant ( P < 0.05).
More axons were lacking a Schwann cell in- be the site at which nociception is initiated.
vestment in tissue fixed with low molarity
Axon to axon contact is low in the inferior albuffer. The proportion of axons exposed veolar nerve (Holland, 1981) but more than a
declined linearly with increasing buffer molar- third of pulpal axons are apposed, a proportion
ity ( P < 0.05). The regression plot of this par- apparently unaffected by the molarity of the
ameter is shown in Figure 4 (upper line). For buffer in the fixative mixture. Coupling becomparison the data derived from our earlier tween pulpal axons has been demonstrated
study on the inferior alveolar nerve of the electrophysiologically (Matthews and Hol-
The proportions of the axons that were
closely apposed to other axons.
The proportions of the axolemma that was
apposed to other axons. The data obtained
was examined statistically. The relationship
of each measured parameter to the buffer
molarity was examined by regression analysis.
474
G.R. HOLLAND
475
BUFFER MOLARITY EFFECT ON AXONS I N RAT MOLAR PULP
TABLE 2. The relationship between buffer molarity and axon size, exposure, and contact,
a s determined bv remession analysis
Buffer molarity vs.
Mean axon circumference
Proportion of axons exposed
Proportion of total axolemma circumference exposed
Proportion of axons apposed
Proportion of total axolemma circumference apposed
Slope
-1.4
-40.2
-21.4
-1.60
-2.01
Standard
error of slope
0.55
4.88
1.82
15.95
16.76
Significance
< 0.05
<0.05
<0.01
N.S.
N.S.
land, 1975)and it has been suggested that this
may occur via gap junctions that link axons
either directly or via an intermediate odontoblast (Matthews and Holland, 1975; Holland,
1975, 1976, 1977, 1980b). Attempts using autoradiography (Byers, 1977)and serial sectioning at the ultrastructural level (Holland,
1980a) have failed to confirm this hypothesis.
The suggestion that mere apposition may result in the spread of nervous activity in this
situation (Holland, 1980a)is supported by the
findings of this study. Although some apposi- M
tion does occur in the more central nerve fibers
it would appear to be relatively insignificant in
amount. Gasser (1955)computed that apposition would have to be very extensive to allow
nervous activity to cross from one axon to another. I t would seem that this degree of con0
1
2
3
4
tact may be reached in the dental pulp, alBuffer Molarily
though, as Bennet (1977) has pointed out,
Fig. 5. Regression analysis of the proportion of axons exmuch would depend on the permeability propposed against buffer molarity. The upper line is for pulpal
erties of the apposed membranes. As axonal nerve fibers, the lower line is for fibers in the inferior alveosize in the dental pulp but not in the inferior lar nerve. Both slopes are significant a t P < 0.05. The data
alveolar nerve (Holland, 1981)is related to buf- for the lower line are from an earlier study (Holland. 1980).
fer molarity, it may be that the membrane
properties of axons in these two sites do differ
potent source of intensely painful sensory exconsiderably.
In the dental pulp there are two anatomical periences.
characteristics - extensive divestment of axACKNOWLEDGMENTS
ons by their Schwann cells and widespread axI would like to thank Susan Pylypas and Linoaxonal apposition - that could form the
basis for the initiation and rapid spread of ner- da Nahnybida for their expert technical assistance, Carolyn Klassen for typing the manuvous activity. As the predominant innervation
script, and Roy Simpson and Glenn Reid for
of the pulp is the A delta and C fiber range
(Beasley and Holland, 1978) and these fiber preparing the illustrations. Cindy Wong advissizes play a special role in pain perception (Mel- ed on the statistical treatment of results. This
study was supported by grant No. MA6468
zack and Wall, 1965),this arrangement could
from the Medical Research Council of Canada.
in part explain why the dental pulp is such a
t
Figs. 1-4. Nonmyelinated pulpal nerve fibers fixed in mixtures containing buffers of different molarity
Fig. 1. 0.05 M, arrow indicates axon of low electron density.
Fig. 2. 0.2 M.
Fig. 3. 0.3 M.
Fig. 4. 0.4 M.
G.R. HOLLAND
476
LITERATURE CITED
Beasley, W.L., and G.R. Holland (1978) A quantitative
analysis of the innervation of the pulp of the cat’s canine
tooth. J . Comp. Neurol., 178:487-494.
Bennet. M.V.L. (1977)Electrical transmission: A functional
analysis and comparison of chemical transmission. In:
Handbook of Physiology. Section I. The Nervous System.
J.M. Brookhart and V.B. Mountcastle. eds. American
Physiological Society, Bethesda, pp. 357-416.
Byers, M.R. (1977)Fine structure of trigeminal receptors in
rat molars. In: Pain in the Trigeminal Region. D.J. Anderson and B. Matthews, eds. Elsevier, New York, pp 37-48.
Gasser, H.S. (1955) Properties of dorsal root unmedullated
fibers on two sides of the ganglion. J. Gen. Physiol.. 32:
709-728.
Holland. G.R. (1975) Membrane junctions on cat odontoblasts. Arch Oral Biol.. 20:551-552.
Holland, G.R. (1976) Lanthanum hydroxide labelling of gap
junctions in the odontoblast layer. Anat. Rec., 186:121126.
Holland, G.R. (1977) Structural relationships in the odonto-
blast layer. In: Pain in the Trigeminal Region. D.J. Anderson and B. Matthews, eds. Elsevier, New York. pp. 25-35.
Holland. G.R. (1980a)Non-myelinated fibers and their terminals in the sub-odontoblasticplexus of the feline dental
pulp. J. Anat., 130:457-467.
Holland, G.R. (1980b)Microtubule and microfilaments populations of cell processes in the dental pulp. Anat. Rec.,
198.421-426.
Holland, G.R. (1981)The extent of axonal exposure and axoaxonal apposition in the non-myelinated nerve fibers of
peripheral nerve trunks and their dependence on buffer
molarity. J. Anat., in press.
Matthews, B., and G.R. Holland (1975) Coupling between
nerves in teeth. Brain Res., 98t354-358.
Melzack, R., and P.D. Wall (1965)Pain mechanisms: A new
theory. Science, 150r971-979.
Ochoa. J. (1976) The unmyelinated nerve-fiber. In: The
Peripheral Nerve. D.N. Landon, ed. Chapman and Hall,
London, pp. 106-158.
Warshawsky, H., and G. Moore (1967) A technique for the
fixation and decalcification of rat incisors for electron microscopy. J . Histochem. Cytochem., 15:542-549.
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effect, exposure, axonal, molar, axoaxonal, molarity, pulp, rat, apposition, buffer
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