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Quantitative ultrastructure of intradental nerve fibres in marmosets
Archs oral Bid. Vol. 17, pp. 645-660, 1972. Pergamoo Press.Printedin GreatBritain.
QUANTITATIVE ULTRASTRUCTURE OF INTRADENTAL NERVE FIBRES IN MARMOSETS K. W. BUELTMANN,* U. L. KARLSSON and J. EDIE Dental Research Laboratory, College of Dentistry and Department of Anatomy, College of Medicine, University of Iowa, Iowa City, Iowa 52240 U.S.A. Summary-Numbers, axon circumferences, myelin widths, nerve investments and in&a-axonal organization were investigated quantitatively for myelinated and unmyelinated nerve fibres in apical cross-sections of adult marmoset tooth pulps. The pulps were preserved in siru by aldehyde perfusion and processed for electron microscopy. Numbers of unmyelinated exceeded the myelinated nerve fibres and their circumference size distributions overlapped by less than 5 per cent. Many unmyelinated fibres were partially or totally non-invested with correspondingly smaller dimensions. Neurotubules and neurofilaments were observed to possess particular and size-dependent geometrical configurations. These results should help establish a quantitative ultrastructural basis for the nerve fibre organization within the tooth pulp.
INTRODUCTION
of a nerve is reflected in the anatomical characteristics of its neuron processes. For instance, both action-potential propagation velocity and functional mode are known to be dependent upon the nerve dimensions and type of axon investment (i.e. GANONG, 1967). There exist many conflicting reports concerning the physiology of the intradental innervation. Although the presence of unmyelinated fibres in the dental pulp has been established (MATTHEWS, DORMAN and BISHOP, 1959; UCHIZONO and HOMMA, 1959; ENGSTR~~M and OHMAN, 1960; FRANK, 1966), there has been no electrophysiologic evidence of C-fibre content (Funakoshi and Zotterman, 1963; NEIDLE and LIEBMAN, 1964; DELANGE, HANNAM and MATTHEWS, 1969). There is much discussion as to whether the intradental nerve is limited in its sensory perception to pain alone or may also be discriminately receptive to heat and cold stimuli (FUNAKOSHI and ZOTTERMAN, 1963; SCOTT and TEMPEL, 1964; MATTHEWS, 1968). In addition, basic disagreements have persisted after many years regarding the interpretation of nociceptorinduced action-potentials within the intradental nerve. In summary, there exist many inadequacies in present structure-function concepts regarding the tooth pulp. Hitherto unknown amounts of participating unmyelinated nerve fibres are conceived important in propagating autonomic as well as pain impulses. Structural similarities and/or differences to other peripheral nerves must also be reconciled for functional interpretation. THE PHYSIOLOGY
* Present Address: University of Southern California, School of Dentistry, Department of Periodontics, Los Angeles, California 90007, U.S.A. 645
K. W. BUELTMANN, U. L. KARLSSON ANDJ. EDIE
646 The primary
aim of this study was, therefore,
to establish
a quantitative
ultra-
structural basis for the nerve fibres entering the tooth. This would provide a fundamental basis for structural studies of the preterminal and terminal characteristics of these same nerves. As a preliminary
to such investigations,
this study also provides
some semiquantitative data on the assumed preterminal portions of these nerves. The significance is that the relative amounts of participating nerve fibres would become known for the first time. Thus far the structural analysis distribution
of the myelinated
of the intradental
nerve has involved
nerve fibres (e.g. GRAF and BJ~~RLIN, 1951;
the size GRAF and
HJELMQUIST, 1955). In these light microscopic studies, the small unmyelinated fibres could not be visualized. UCHIZONO and HOMMA (1959) measured a limited number of unmyelinated
fibres within the human pulp by electron microscopy,
enumeration of participating nerve fibres. The direct diameter measurements as performed errors
since they imply cylindrical
shapes
but did not attempt
in previous studies may introduce
of nerve fibres.
Measurements
of nerve
dimensions must fulfill several criteria in order to be useful. For example, they should (1) represent a parameter pertinent to electric-potential propagation characteristics, (2) permit
comparison
between
fibre shape independence in vivo and (4) if possible, appeared ference
that the parameter that has previously
the present
myelinated
and unmyelinated
fibres,
(3) possess
a
since it is not known what shape a nerve fibre assumes permit comparison with the results of previous studies. It which best fulfills the above criteria was the axon circumbeen used by FRIEDE and SAMORAJSKI (1967).
study attempted
to obtain
nerve fibre circumference
Therefore,
distributions
for all
myelinated and most unmyelinated nerve fibres for the marmoset intradental nerve. In addition, the intra-axonal organization and axonal investment are described since it may reflect upon the specific functions
MATERIALS
of this nerve.
AND
METHODS
Seven mandibular incisors and 2 mandibular canine teeth from 6 adult cotton-top marmosets (Sug&zus oedipus) were used for the study. Each of the 4 males and 2 females was apparently healthy and possessed the complete adult dentition (2 incisers, 1 canine, 3 premolars and 2 molars in each quadrant) as described by GREGORY(1922). Their weights ranged from 350 to 465 g. Each animal was injected intraperitoneally with 10,000 USP units of isotonic aqueous sodium heparin. They were initially sedated with an intramuscular injection of 0.8 mg of phencyclidine hydrochloride. Surgical anaesthesia was obtained by intraperitoneal injection of 25 mg sodium pentobarbitol per kilogram of body weight. Fixation of the pulp was accomplished irz situ by a modification of the aldehyde perfusion procedure described by KARLSSONand SCHULTZ(1965). The pre-wash amounted to approximately 30 ml of the 320 mOsM phosphate buffer. After the perfusion, the extracted teeth were scored with a diamond disk in such a manner as to avoid overheating. The pulps were subsequently exposed by carefully cracking the teeth in a vice. The apical part of the pulps was carefully dissected free, rinsed in the phosphate buffer and postfixed in 1 per cent buffered osmium tetroxide for 2-4 hr. After another short buffer rinse, the specimens were dehydrated in increasing concentrations of acetone and embedded in Vestopal W. Thin sections for electron microscopy, cut at 650-1000 A, were obtained 1-2 mm from the apical foramen. The obliquity of the section planes were estimated to deviate by less than 5” from the plane normal to the nerve axis. The sections were collected on Formvar-supported single-hole grids and
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stained at room temperature, first with a saturated solution of many1 acetate for 1 hr and then for 1 min with lead citrate. The specimens were observed in a Siemens 101 electron microscope. Twoseries of micrographswere taken, the first at x 2400 magnification for myelinated fibres and a second at x 10,000 magnification for unmyelinated fibres. The magnification of the microscope was calibrated with a replica carbon grating. Occasional survey ( x 240) and higher magnification ( x 20,000) micrographs were taken for recording features of the nerve. All negatives were magnified 3 times. The recordings consisted of measuring the axon circumferences of both fibre types with a linear measuring wheel which had a precision of measurement of & O-2 cm (0.3 pm and 0.07 pm for myelinated and unmyelinated circumferences respectively). An equivalent circular fibre-diameter was estimated by dividing this measurement by rr. The portion of each unmyelinated profile circumference which was not invested in Schwann cell membrane was measured. The thickness of the myelin sheath was noted for each myelinated profile using a measuring magnifier calibrated to * 0.1 mm (precision of measurement O-05 mm N 0.08 pm). Prior to tabulation, the individual nerve profiles were classified as either being contained within the primary core of nerve fibres (“core”) or between that and the odontoblastic layer (“periphery”). Nerve fibres not in immediate contact with those of the core were considered as peripheral fibres (see Fig. 1). A limited number of micrographs (at x 60,000 magnification) were selected for examining the intra-axonal organization of both the myelinated and unmyelinated profiles, In each nerve profile, the circumference was noted and the number of enclosed tubules and filaments counted. The data were recorded on computer punch cards and analysed on an IBM 360/65 computer for determination of means, standard deviations, frequency distributions and correlations. RESULTS
Qualitative observations All specimens demonstrated adequate membrane and organelle integrity from the ultrastructural point of view. Collagen and connective tissue elements filled the extracellular spaces and obvious empty spaces were absent. Among the nervous elements, a rather distinct pattern of distribution was seen in all specimens. Near the centre of the pulp, a condensation of myelinated and unmyelinated profiles was observed (Fig. 1). Outside, or peripheral, to this central condensation or “core”, some groups or single myelinated profiles were always observed as were the more numerous unmyelinated profiles. The cross-sectioned core exhibited all the ultrastructural characteristics of a peripheral nerve, including myelinated profiles, nodes of Ranvier, Schwann cell nuclei, fibroblast profiles, unmyelinated profiles and collagen bundles (Fig. 2). However, the core did not demonstrate the usual peri- and epineural sheaths. In only one of the specimens was a tendency towards complete endoneural ensheathment observed (Fig. 2). All the others had scattered fibroblastic cell bodies among the nerve profiles that otherwise were surrounded by collagen filaments up to about 1000 A in diameter. The most notable feature in all the specimens was the variety of shapes assumed by the myelinated profiles and the wide variation in the width of their myelin sheaths (Fig. 2). The sheaths were generally intact and the regularly layered myelin demonstrated a periodicity of about 1000 A. A negligible amount of myelin sheath splitting occurred in the regions of the incisures and the nodes of Ranvier. Inner and outer mesaxons were often distinguishable. As expected, most unmyelinated profiles were grouped and invested in Schwann cell processes (Fig. 3). However, some profiles were found to be partially invested or
648
K. W. BUELTMANN, U. L. KARLSSON AND J. EDIE
surrounded only by basement membrane material (Fig. 4) and were differentiated from the round profiles of other types of cell processes by their characteristic cytoplasmic organization (see below). A few partially invested profiles were observed to appose each other without intervening material. The nerve cytoplasm was characterized by typical neurotubules, neurofilaments, mitochondria and small membrane-bound vesicles (Fig. 3). Only neurotubules were seen in the smallest profiles. In larger profiles, the neurotubules assumed a peripheral position and the filaments appeared as a packet filling in the central region. In still larger profiles, vesicles appeared among the peripheral neurotubules and, whenever larger organelles were observed, they were often surrounded by neurotubules. This pattern could be distinguished in all specimens. A similar pattern was observed in myelinated profiles (Fig. 5). The arrangement often suggested that a characteristic minimum distance between organelles is maintained. Quantitative aspects of this organization follow below. Neither the membranes of the myelinated nor unmyelinated profiles were ever observed in contact with the endothelial cells lining the vessels. Rather there was always an interposed layer of collagen filaments, smooth muscle cells and occasionally the cell processes of a fibroblast. Quantitative observations A summary of the data according to the source, location and the presence of myelin is presented in Table 1. All nerve profiles, with the exception of most of the unmyelinated profiles in the periphery, were examined.
Myelinated nerve fibres
No. of specimens Total No. Mean No./spccimen Range
Unmyelinated nerve fibres No. of specimens Total No. Mean No./specimen Range
2 192 96 55-137
697 116 53-230
2 260
6 839
130 105-155
140 50-22s
6
2 79 39 3841
2 86 43 9-77
2 773 (sample)
3 211 (sample)
382-391
45-121
At the apical level of the pulp, 55 per cent of the total number the core were unmyelinated. When all nerve profiles within a pulp considered, the number of unmyelinated profiles were estimated to that of the myelinated. Since the odontoblastic layer was absent
of profiles within cross-section were be at least double from parts of the
QUANTITATIVE
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pulp cross-section (Fig. 1), it was impossible to account for the total number of unmyelinated fibres. The distribution of axon circumferences in the core and periphery of the intradental nerve for myelinated and unmyelinated profiles is shown in Fig. 6. The unmyelinated and myelinated profiles showed peaks at 1 pm and 8 pm circumference, respectively. The data produced on overlap between the unmyelinated and myelinated distributions of less than 5 per cent of the total number of fibres. Number UM .
l
o----.
Core Periphery
Circumference I 0
I
2 Equivalent
in 3
diameter
I099
I64
urn 4
in
984
M 889
5
6
urn
FIG. 6. Distributions for core (solid line) and peripheral (dashed line) axon circumferences of unmyelinated (UM) and myelinated (M) nerve profiles are displayed, the class interval widths were 0.27 pm and l-11 pm for unmyelinated and myelinated nerves, respectively. The “equivalent diameter” represents that diameter a circular nerve profile of the same circumference would possess.
The myelin sheaths in the core had an average width of 0.26 If 0.08 pm. The mean proportion of sheath width to axon circumference was 31 f. 13 per cent. The myelinated profiles found in the pulp outside the intradental nerve core had myelin sheaths averaging 0.25 -& 0.07 pm thick. The mean ratio of sheath thickness to circumference was 31 * 15 per cent for the peripheral fibres. The product moment correlation of myelin sheath width to the circumference of the same nerve profile was tested on 1053 profiles. The range of correlation coefficients in all specimens was - O-174 to + O-235 with a mean of 0’ 134. A.O.B. 17/4-c
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K. W. BUELTMANN, U. L. KARLSSON AND J. EDIE
The number of myelinating Schwann cell nuclei per cross-section ranged from 6 to 38 in 8 specimens observed. The number was fairly well correlated to the amount of myelinated profiles in each specimen with an average of one nucleus seen for every 7.5 myelinated profiles. Only an average of one node of Ranvier per 24 myelinated profiles was observed. There was a wide range (1-14) of nodes per specimen. Scatter diagrams for number of neurotubules and neurofilaments versus axon circumference are illustrated in Figs. 7 and 8, respectively. The method of least squares was used to fit polynomial curves to the data. The best curves and their equations are displayed on the scatter diagrams. The size distribution of unmyelinated profiles fully invested by Schwann cell cytoplasm is compared to the distributions of partially and completely non-invested unmyelinated profiles in Fig. 9. Of the 2083 unmyelinated profiles analysed, about 50-
. .
45T = (16.8
40-
ym-1)
C -
6.3 .
r = 0.786 . .
35-
. l.
30-
. /
c
. .
l
.
g
. .
. .
.
IO-
.
l
5-
.:
.
.
. l
.
l
. *
*as 8 l . . . ..
l e8 . . . i//-/
o0
.
.
. .
.
.
a .
.
.
20
I.0 Circumference
(C)
in
ym
FIG. 7. Scatter diagram for number of neurotubules per nerve profile versus the axon cross-section circumference. The method of least squares yields the straight line as the best fit to the data.
QUANTITATIVEORGANLZATIONOFINTRADENTALNERVEINMARMOSET
651
300-
. 250_
F = (39.6 p&) C* - (II.2
pm-l)
C + 73
. l
.
2 ,200z E 0 = + 0
150-
L _z 5 z
IOO-
50-
20
I.0 Circumference
in
urn
FIG.8. Scatter diagrams for number of neurofilaments per nerve profile vs. the axon cross-section circumference. The method of least squares yields the parabola as the best fit to the data.
30 per cent were only partially invested by Schwann cells.
invested
while about
4 per cent were completely
non-
DISCUSSION
The subdivision of fibres into “core” and “periphery” was only based on the definition of the group of fibres constituting the core (Fig. 1). The basis for subdivision was therefore arbitrary but served the interpretative purpose of distinguishing between fibres entering the tooth and destined to more coronal portions of the pulp (core fibres) from those following the vasculature or those preterminal fibres innervating the odontoblasts at or near that cross-section. More unmyelinated than myelinated nerve fibres were consistently observed within the intradental nerve cores. This large amount of unmyelinated nerve fibres entering the tooth must undoubtedly have a major contribution to the physiological processes within it. These results are in conflict with previous publications that report fewer unmyelinated fibres (WINDLE, 1927; GRAF and BJBRLIN, 1951; UCHIZONO and HOMMA, 1959; PISCHINGER and STOCKINGER, 1968).The difference is due to the inability of the light
652
K. W. BUELTMANN,U. L. KARLSSONAND J.
EDIE Number
.
.
Completely
89
non-invested
._______, Partially
596
invested +.
_.
-.
Completely
2 Circumference I 0.5 Equivalent
1398
in
Am
I I.0 diameter
in
yrn
FIG. 9. Fibre circumference distributions of fully invested (solid and dashed line) partially invested (dashed line) and non-invested (solid) nerve profiles. The greater the degree of
investment, the more likely the nerve profile will assume large circumferences. The class interval width was 0.27 pm. microscopic and previous histologic techniques to observe adequately and count the unmyelinated fibres. In this investigation, all the observed myelinated fibres were counted and measured. Very few escaped detection as only parts of the odontoblastic layer was missing from each specimen. The number of nerve fibres per tooth is comparable to that obtained from human teeth by GRAF and BJ~~RLIN (1951). With all the in-vitro criteria for nervous tissue preservation fulfilled (CAULFIELD, 1957; ROBERTSON, 1960; SJ~~STRAND,1967; BERTHOLD, 1968), both the unmyelinated and, in particular, the myelinated fibres were found to have irregular, rather than circular, contours. BERTHOLD(1968) suggested that this irregularity in form is a direct reflection of their in-vivo shape which results from the myelin sheath adapting its shape to localized
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accumulations of the surrounding Schwann cell cytoplasm. If this view is acceptable, the circular profiles resulting from previous preservation methods must be deemed artifactual. A comparison of previous diameter measurements with the circumference measurements of this investigation is possible if it is assumed that the axon circumference retains the same length for both electron and light microscopic preservation methods. Then, the comparable diameters (equivalent axon diameters, C/n, plus twice the average myelin sheath widths) from this investigation are, on the average, 0.5 pm larger than those found by GRAF and BJ~RLIN(1951). There was no apparent difference in the size of the myelinated fibres in the core and periphery (Fig. 6). The increased measurement precision and relative shape independence using electron microscopic techniques should permit more reliable results. However, the validity of any geometrical measurement must await the establishment of the true in-vivo shape of nerve fibres. The unmyelinated fibre-size distribution (Fig. 6) conflicts with that reported by UCHIZONOand HOMMA(1959). Their unimodal diameter distribution had a range from 0 *5 to 2 *5 pm, a modal value of about 1 *Opm and an overlap with the myelinated spectra from 1 .O to 2 *5 I_cm(including 45 per cent of all fibres). The present study shows a much smaller overlap (less than 5 per cent), a range of circumference measurements from 0.8 to 1.6 pm (equivalent diameters from 0.25 to 0 *50 pm) and a modal value of about 1 .O pm (0.3 pm equivalent diameter). It is unlikely that these discrepancies can be attributed to species differences and different processing methods alone. The cross-sectioned core exhibited all the ultrastructural characteristics of a peripheral nerve. Only the lack of fasciculation and a nerve sheath were observed. This may be reflected functionally in an increased sensitivity of the nerve to mechanical stimuli. Indeed, pulsating electrophysiologic responses, as originating from pulpal vessels, have been recorded (FUNAKOSHIand ZOTTERMAN,1963). One may even question the nerve ending per se as being the only location for stimulus since naked nerve fibres, such as those observed here (Fig. 4), may be very sensitive to environmental changes. If true, the functional concept of the nerve ending may then be extended to the preterminal part of the intradental nerve. This view would have to be reconciled with those discussed in recent publications (EIFINGER,1969; S~OCKINGER and PRITZ, 1970). The presence of non-invested nerve fibres does not necessarily imply that unmyelinated fibres are non-invested along their entire course since this was a two-dimensional analysis. They may actually represent “nodes” of unmyelinated fibres since they are thinner than the invested profiles (Fig. 9). An apparent corroboration exists in that nodes of the myelinated fibres appear with the same relative frequency in our material. This interpretation raises a question about the exact mode of the propagation mechanism in unmyelinated nerves. The data correlating neurofilament and neurotubule numbers to axon circumference and area, respectively, appears to be a quantitative and geometrical account of observations noted by PISCHINGERand STOCKINGER,(1968). They noted that the smaller axon profiles contained mostly filaments.
654
K. W. BUELTMANN,U. L. KARLSSONANDJ. EDIE
The apparent proportionality between the number of neurotubules and axon circumference suggests the neurotubules are arranged in curvilinear patterns. It appeared that the main pattern was a circle concentric with the axon axis but with some tubules surrounding larger organelles. There were, on the average, 17 additional tubules for each micrometer circumference increment above 0.4 pm. On the other hand, the parabolic relationship between the number of neurofilaments and axon circumference suggests the neurofilament number is dependent upon axon cross-sectional area and that the neurofilaments are distributed with uniform density within the axon. An examination of the micrographs reveals that the distribution excludes the periphery of the axon’s interior. There were, on the average, 40 additional filaments for every additional square micrometer of axon cross-sectional area. Acknowledgements-Supported by intramural funds from the Graduate College, Department of Periodontology, Dental Research Laboratory, from the NIH General Research Support Grant award to College of Dentistry and from the Neurosensory Center, Program Project Grant Number NSO 3354 of the National Institute of Neurological Diseases and Stroke. R&umb-On a investiguC quantativement le nombre, les circonfbrences des axons, les quantites de mykline, les couvertures desnerfs et l’organisation intra-axionale, dans des sections transversales apicales de la pulpe de la dent chez les marmottes, pour les fibres nerveuses mytlinCes et non-mytlinCes. Les pulpes ont 6tB preservees in situ par perfusion d’aldkhyde et prtparkes pour la microscopic Clectronique. Le nombre des fibres nonmytlinCes dCpassait les fibres my6lin6es, la difft%ence dans les distributions de leurs dimensions ttant moindre que 5 pour cent. Des nombreuses fibres non-myClinCes Ctaient partiellement ou totalement non-couvertes et cons&quemment d’une dimension plus rCduite. On a remarque des neurotubules et des neurofilaments ayant des configurations gComttriques particuli&es, dependant de leur dimension. Ces rtsultats devraient aider ?I Ctablir une base ultrastructurale quantitative pour l’organisation de la fibre nerveuse B I’int&ieur de la pulpe de la dent. Zusammenfassung-Es wurden eine Anzahl von Neuritenumfgngen, Myelinbreiten, Nervenumhiillungen und interaxonaler Organismus mengenmgssig fiir myelinisierte und nicht myelinisierte Nervenfasern in Apikalquerschnitten der Pulpa des ausgewachsenen Krallenaffen untersucht. Die Pulpas wurden in der natiirichen Lage durch Ubergiessen mit Aldehyd prgserviert und ffir Elektromikroskopie behandelt. Die Zahlen der nicht myelinisierten Fasern iiberschritten die myelinisierten Nervenfasern und die Umfangsverzweigungen iiberlappten in Grijsse urn weniger als 5 Prozent. Viele, nicht myelinisierte Fasern waren mit entsprechender kleineren Massen teilweise oder vdllig nicht umhiillt. NeurokanPlchen und Fiserchen besassen nach der Beobachtung besondere und grtissenabhlngige, geometrische Bildungen. Diese Resultate sollten dazu beitragen, eine mengenmgssige, ultrastrukturelle Grundlage fiir den Nervfaserorganismus innerhalb der Zahnpulpa festzulegen. REFERENCES BERTHOLD,C. H. 1968. Ultrastructure of the node-paranode region of the mature feline ventral lumbar spinal-root fibres. Acta Sot. Med. Upsal. 73,37-67. CAULFIELD,J. B. 1957. Effects of varying the vehicle for 0~0, in tissue fixation. J. Biophysic. Biochem. Cytol. 3, 827-830.
DELANGE, A., HANNAM,A. G. and MATTHEWS,B. 1969. The diameters and conduction velocities of fibres in the terminal branches of the inferior alveolar nerve. Archs oral Biol. 14,513-519.
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EIFINGER, F. 1969. Zum Problem der Pulpa-Dentin-Innervation. Dt. Zahn-, Mund- Kieferheilk., B6 58, H 5/S, 188-201. ENGSTR~M,H. and &MAN, A. 1960. Studies on the innervation of human teeth. J. dent. Res. 39, 799-809. FRANK, R. M. 1966. Ultrastructure of human dentine. In: Third Europ. Symp. on Calcified Tissues (edited by FLEISH,BLACKWOODand OWEN), pp. 259-272. Springer, Berlin. FRIEDE, R. L. and SAMORAJSKI, T. 1967. Relation between the number of myelin lamellae and axon circumference in fibers of vagus and sciatic nerves of mice. J. camp. Neurol. 130-131,223-231. FUNAKOSHI,M. and ZOTTERMAN, Y. 1963. A study in the excitation of dental pulp nerve fibres. In: Sensory Mechanisms in Dentine. (Edited by ANDERSON,D. J.) pp. 60-72. Pergamon Press, Oxford. GANONG, W. J. 1967. In: Review of Medical Physiology, p. 32. Lange Medical Publications, Los Altos. GRAF, W. and BJORLIN,G. 1951. Diameters of nerve fibers in human tooth pulps. J. Am. dent. Ass. 43,186193.
GRAF, W. and HJELMQUIST,U. 1955. Caliber spectra of dental nerves in dogs and cattle. J. camp. Neurol. 103,345-353. GREGORY,W. K. 1922. The Origin and Evolution of the Human Dentition, pp. 218-219, Baltimore, Maryland. KARLSSON,U. and SCHULTZ,R. 1965. Fixation of the central nervous system for electron microscopy by aldehyde perfusion. J. Ultrastruct. Res. 12, 160-186. MATTHEWS,B. 1968. Cold-sensitive and heat-sensitive nerves in teeth. J. dent. Res. 49, 974-975 (Abstract). MATTHEWS,J. L., DORMAN,H. L. and BISHOP,J. G. 1959. Fine structure of the dental pulp. J. dent. Res. 38, 940-946. NEIDLE,E. A. and LIEBMAN,F. M. 1964. Effects of vasoactive drugs and nerve stimulation on blood flow in the tooth pulp and allied structures of the cat. J. dent. Res. 45,412422. PISCHINGER,A. and STOCKINGER, L. 1968. Die Nerven der menschlichen Zahnpulpa. Z. Zelfjbrsch. 89, 44-61. ROBERTSON,J. D. 1960. The molecular structure and contact relationships of cell membranes. Prog. Biophys. 10, 343418. SCOTT,D. JR. and TEMPEL,T. 1963. Receptor potentials in response to thermal and other excitation. In: Sensory Mechanisms in Denrine (Edited by ANDERSON,D. J.). Oxford, Pergamon Press. SJ~STRAND,F. S. 1967. Electron Microscopy of Cells and Tissues, Vol. l., p. 410, Academic Press, New York. STOCKINGER,L. and Pturz, W. 1970. Morphologische Aspekte der Schmerzempfindung im Zahn. Dr. Zahn. Z. 25(5), 557-565. UCHIZONO,K. and HOMMA,K. 1959. Electron microscopic studies on nerves of human tooth pulp. J. dent. Res. 38, 1133-l 151. WINDLE, W. F. 1927. The distribution and probable significance of unmyelinated nerve fibres in the trigeminal nerve of the cat. J. camp. Neurol. 41,453-477.
PLATES1-5
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PLATE 1 FIG. 1. A cross-section of dental pulp close to the apex of a marmoset incisor tooth. The rectangular outline encloses the bundle of nerve fibres referred to as “core “fibres. Those fibres beyond this outline and with no obvious connection to the core fibres constitute the “periphery”. O-5 pm section, methylene blue. x 80
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PLATE 2
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PLATE 2 FIG. 2. Survey electron micrograph of the intradental nerve core that is preserved by glutaraldehyde/OsO, fixation. Note the irregular contours of the myelinated profiles, the variation of myelin sheath thickness and the groups of unmyelinated profiles (U). It was necessary to examine the unmyelinated profiles at higher magnifications before their true identities could be established. In the lower half. a fibroblast process @) is traversing the core. A nucleated Schwann cell (S) and a profile sectioned close to a node of Ranvier (R) can be seen. x 10,300
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PLATE 3 FIG. 3. A group of unmyelinated profiles are seen surrounded by variable amounts of a Schwann cell process. The Schwann cell cytoplasm (S) is more opaque than the
axoplasm, which displays a typical organization of neurotubuIes (T) at the periphery and neurofilaments (F) in the interior of the larger profiles. The nerves are separate from the investing Schwann cell cytoplasm by a SO-200 A space. x 72,000
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PLATE 4 FIG. 4. A non-invested nerve profile is displayed below. It is not invested by Schwann cell membrane, but is surrounded by basement membrane material (BM). The profile is identified as a nerve process since it exhibits the characteristic neurotubule/neurofilament organization. Above is seen part of a myelin sheath with surrounding Schwann cell invested by basement membrane material (BM). x 184,000
659
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PLATE 5
FIG. 5. A myelinated profile is seen sectioned in its para-nodal region. Note the diikrence in the opacities of the Schwann cell cytoplasm (S) and the axoplasm (A). The neurotubules (T) and vesicles (V) are arranged along the periphery of the nerve process and around mitochondria (M). Filaments (F) cover the remaining surface. The opaque granular material in the Schwann cytoplasm was typically found in the para-nodal regions of the fibre. (About x 100,000)
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QUANTITATIVE ULTRASTRUCTURE OF INTRADENTAL NERVE FIBRES IN MARMOSETS K. W. BUELTMANN,* U. L. KARLSSON and J. EDIE Dental Research Laboratory, College of Dentistry and Department of Anatomy, College of Medicine, University of Iowa, Iowa City, Iowa 52240 U.S.A. Summary-Numbers, axon circumferences, myelin widths, nerve investments and in&a-axonal organization were investigated quantitatively for myelinated and unmyelinated nerve fibres in apical cross-sections of adult marmoset tooth pulps. The pulps were preserved in siru by aldehyde perfusion and processed for electron microscopy. Numbers of unmyelinated exceeded the myelinated nerve fibres and their circumference size distributions overlapped by less than 5 per cent. Many unmyelinated fibres were partially or totally non-invested with correspondingly smaller dimensions. Neurotubules and neurofilaments were observed to possess particular and size-dependent geometrical configurations. These results should help establish a quantitative ultrastructural basis for the nerve fibre organization within the tooth pulp.
INTRODUCTION
of a nerve is reflected in the anatomical characteristics of its neuron processes. For instance, both action-potential propagation velocity and functional mode are known to be dependent upon the nerve dimensions and type of axon investment (i.e. GANONG, 1967). There exist many conflicting reports concerning the physiology of the intradental innervation. Although the presence of unmyelinated fibres in the dental pulp has been established (MATTHEWS, DORMAN and BISHOP, 1959; UCHIZONO and HOMMA, 1959; ENGSTR~~M and OHMAN, 1960; FRANK, 1966), there has been no electrophysiologic evidence of C-fibre content (Funakoshi and Zotterman, 1963; NEIDLE and LIEBMAN, 1964; DELANGE, HANNAM and MATTHEWS, 1969). There is much discussion as to whether the intradental nerve is limited in its sensory perception to pain alone or may also be discriminately receptive to heat and cold stimuli (FUNAKOSHI and ZOTTERMAN, 1963; SCOTT and TEMPEL, 1964; MATTHEWS, 1968). In addition, basic disagreements have persisted after many years regarding the interpretation of nociceptorinduced action-potentials within the intradental nerve. In summary, there exist many inadequacies in present structure-function concepts regarding the tooth pulp. Hitherto unknown amounts of participating unmyelinated nerve fibres are conceived important in propagating autonomic as well as pain impulses. Structural similarities and/or differences to other peripheral nerves must also be reconciled for functional interpretation. THE PHYSIOLOGY
* Present Address: University of Southern California, School of Dentistry, Department of Periodontics, Los Angeles, California 90007, U.S.A. 645
K. W. BUELTMANN, U. L. KARLSSON ANDJ. EDIE
646 The primary
aim of this study was, therefore,
to establish
a quantitative
ultra-
structural basis for the nerve fibres entering the tooth. This would provide a fundamental basis for structural studies of the preterminal and terminal characteristics of these same nerves. As a preliminary
to such investigations,
this study also provides
some semiquantitative data on the assumed preterminal portions of these nerves. The significance is that the relative amounts of participating nerve fibres would become known for the first time. Thus far the structural analysis distribution
of the myelinated
of the intradental
nerve has involved
nerve fibres (e.g. GRAF and BJ~~RLIN, 1951;
the size GRAF and
HJELMQUIST, 1955). In these light microscopic studies, the small unmyelinated fibres could not be visualized. UCHIZONO and HOMMA (1959) measured a limited number of unmyelinated
fibres within the human pulp by electron microscopy,
enumeration of participating nerve fibres. The direct diameter measurements as performed errors
since they imply cylindrical
shapes
but did not attempt
in previous studies may introduce
of nerve fibres.
Measurements
of nerve
dimensions must fulfill several criteria in order to be useful. For example, they should (1) represent a parameter pertinent to electric-potential propagation characteristics, (2) permit
comparison
between
fibre shape independence in vivo and (4) if possible, appeared ference
that the parameter that has previously
the present
myelinated
and unmyelinated
fibres,
(3) possess
a
since it is not known what shape a nerve fibre assumes permit comparison with the results of previous studies. It which best fulfills the above criteria was the axon circumbeen used by FRIEDE and SAMORAJSKI (1967).
study attempted
to obtain
nerve fibre circumference
Therefore,
distributions
for all
myelinated and most unmyelinated nerve fibres for the marmoset intradental nerve. In addition, the intra-axonal organization and axonal investment are described since it may reflect upon the specific functions
MATERIALS
of this nerve.
AND
METHODS
Seven mandibular incisors and 2 mandibular canine teeth from 6 adult cotton-top marmosets (Sug&zus oedipus) were used for the study. Each of the 4 males and 2 females was apparently healthy and possessed the complete adult dentition (2 incisers, 1 canine, 3 premolars and 2 molars in each quadrant) as described by GREGORY(1922). Their weights ranged from 350 to 465 g. Each animal was injected intraperitoneally with 10,000 USP units of isotonic aqueous sodium heparin. They were initially sedated with an intramuscular injection of 0.8 mg of phencyclidine hydrochloride. Surgical anaesthesia was obtained by intraperitoneal injection of 25 mg sodium pentobarbitol per kilogram of body weight. Fixation of the pulp was accomplished irz situ by a modification of the aldehyde perfusion procedure described by KARLSSONand SCHULTZ(1965). The pre-wash amounted to approximately 30 ml of the 320 mOsM phosphate buffer. After the perfusion, the extracted teeth were scored with a diamond disk in such a manner as to avoid overheating. The pulps were subsequently exposed by carefully cracking the teeth in a vice. The apical part of the pulps was carefully dissected free, rinsed in the phosphate buffer and postfixed in 1 per cent buffered osmium tetroxide for 2-4 hr. After another short buffer rinse, the specimens were dehydrated in increasing concentrations of acetone and embedded in Vestopal W. Thin sections for electron microscopy, cut at 650-1000 A, were obtained 1-2 mm from the apical foramen. The obliquity of the section planes were estimated to deviate by less than 5” from the plane normal to the nerve axis. The sections were collected on Formvar-supported single-hole grids and
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stained at room temperature, first with a saturated solution of many1 acetate for 1 hr and then for 1 min with lead citrate. The specimens were observed in a Siemens 101 electron microscope. Twoseries of micrographswere taken, the first at x 2400 magnification for myelinated fibres and a second at x 10,000 magnification for unmyelinated fibres. The magnification of the microscope was calibrated with a replica carbon grating. Occasional survey ( x 240) and higher magnification ( x 20,000) micrographs were taken for recording features of the nerve. All negatives were magnified 3 times. The recordings consisted of measuring the axon circumferences of both fibre types with a linear measuring wheel which had a precision of measurement of & O-2 cm (0.3 pm and 0.07 pm for myelinated and unmyelinated circumferences respectively). An equivalent circular fibre-diameter was estimated by dividing this measurement by rr. The portion of each unmyelinated profile circumference which was not invested in Schwann cell membrane was measured. The thickness of the myelin sheath was noted for each myelinated profile using a measuring magnifier calibrated to * 0.1 mm (precision of measurement O-05 mm N 0.08 pm). Prior to tabulation, the individual nerve profiles were classified as either being contained within the primary core of nerve fibres (“core”) or between that and the odontoblastic layer (“periphery”). Nerve fibres not in immediate contact with those of the core were considered as peripheral fibres (see Fig. 1). A limited number of micrographs (at x 60,000 magnification) were selected for examining the intra-axonal organization of both the myelinated and unmyelinated profiles, In each nerve profile, the circumference was noted and the number of enclosed tubules and filaments counted. The data were recorded on computer punch cards and analysed on an IBM 360/65 computer for determination of means, standard deviations, frequency distributions and correlations. RESULTS
Qualitative observations All specimens demonstrated adequate membrane and organelle integrity from the ultrastructural point of view. Collagen and connective tissue elements filled the extracellular spaces and obvious empty spaces were absent. Among the nervous elements, a rather distinct pattern of distribution was seen in all specimens. Near the centre of the pulp, a condensation of myelinated and unmyelinated profiles was observed (Fig. 1). Outside, or peripheral, to this central condensation or “core”, some groups or single myelinated profiles were always observed as were the more numerous unmyelinated profiles. The cross-sectioned core exhibited all the ultrastructural characteristics of a peripheral nerve, including myelinated profiles, nodes of Ranvier, Schwann cell nuclei, fibroblast profiles, unmyelinated profiles and collagen bundles (Fig. 2). However, the core did not demonstrate the usual peri- and epineural sheaths. In only one of the specimens was a tendency towards complete endoneural ensheathment observed (Fig. 2). All the others had scattered fibroblastic cell bodies among the nerve profiles that otherwise were surrounded by collagen filaments up to about 1000 A in diameter. The most notable feature in all the specimens was the variety of shapes assumed by the myelinated profiles and the wide variation in the width of their myelin sheaths (Fig. 2). The sheaths were generally intact and the regularly layered myelin demonstrated a periodicity of about 1000 A. A negligible amount of myelin sheath splitting occurred in the regions of the incisures and the nodes of Ranvier. Inner and outer mesaxons were often distinguishable. As expected, most unmyelinated profiles were grouped and invested in Schwann cell processes (Fig. 3). However, some profiles were found to be partially invested or
648
K. W. BUELTMANN, U. L. KARLSSON AND J. EDIE
surrounded only by basement membrane material (Fig. 4) and were differentiated from the round profiles of other types of cell processes by their characteristic cytoplasmic organization (see below). A few partially invested profiles were observed to appose each other without intervening material. The nerve cytoplasm was characterized by typical neurotubules, neurofilaments, mitochondria and small membrane-bound vesicles (Fig. 3). Only neurotubules were seen in the smallest profiles. In larger profiles, the neurotubules assumed a peripheral position and the filaments appeared as a packet filling in the central region. In still larger profiles, vesicles appeared among the peripheral neurotubules and, whenever larger organelles were observed, they were often surrounded by neurotubules. This pattern could be distinguished in all specimens. A similar pattern was observed in myelinated profiles (Fig. 5). The arrangement often suggested that a characteristic minimum distance between organelles is maintained. Quantitative aspects of this organization follow below. Neither the membranes of the myelinated nor unmyelinated profiles were ever observed in contact with the endothelial cells lining the vessels. Rather there was always an interposed layer of collagen filaments, smooth muscle cells and occasionally the cell processes of a fibroblast. Quantitative observations A summary of the data according to the source, location and the presence of myelin is presented in Table 1. All nerve profiles, with the exception of most of the unmyelinated profiles in the periphery, were examined.
Myelinated nerve fibres
No. of specimens Total No. Mean No./spccimen Range
Unmyelinated nerve fibres No. of specimens Total No. Mean No./specimen Range
2 192 96 55-137
697 116 53-230
2 260
6 839
130 105-155
140 50-22s
6
2 79 39 3841
2 86 43 9-77
2 773 (sample)
3 211 (sample)
382-391
45-121
At the apical level of the pulp, 55 per cent of the total number the core were unmyelinated. When all nerve profiles within a pulp considered, the number of unmyelinated profiles were estimated to that of the myelinated. Since the odontoblastic layer was absent
of profiles within cross-section were be at least double from parts of the
QUANTITATIVE
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pulp cross-section (Fig. 1), it was impossible to account for the total number of unmyelinated fibres. The distribution of axon circumferences in the core and periphery of the intradental nerve for myelinated and unmyelinated profiles is shown in Fig. 6. The unmyelinated and myelinated profiles showed peaks at 1 pm and 8 pm circumference, respectively. The data produced on overlap between the unmyelinated and myelinated distributions of less than 5 per cent of the total number of fibres. Number UM .
l
o----.
Core Periphery
Circumference I 0
I
2 Equivalent
in 3
diameter
I099
I64
urn 4
in
984
M 889
5
6
urn
FIG. 6. Distributions for core (solid line) and peripheral (dashed line) axon circumferences of unmyelinated (UM) and myelinated (M) nerve profiles are displayed, the class interval widths were 0.27 pm and l-11 pm for unmyelinated and myelinated nerves, respectively. The “equivalent diameter” represents that diameter a circular nerve profile of the same circumference would possess.
The myelin sheaths in the core had an average width of 0.26 If 0.08 pm. The mean proportion of sheath width to axon circumference was 31 f. 13 per cent. The myelinated profiles found in the pulp outside the intradental nerve core had myelin sheaths averaging 0.25 -& 0.07 pm thick. The mean ratio of sheath thickness to circumference was 31 * 15 per cent for the peripheral fibres. The product moment correlation of myelin sheath width to the circumference of the same nerve profile was tested on 1053 profiles. The range of correlation coefficients in all specimens was - O-174 to + O-235 with a mean of 0’ 134. A.O.B. 17/4-c
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K. W. BUELTMANN, U. L. KARLSSON AND J. EDIE
The number of myelinating Schwann cell nuclei per cross-section ranged from 6 to 38 in 8 specimens observed. The number was fairly well correlated to the amount of myelinated profiles in each specimen with an average of one nucleus seen for every 7.5 myelinated profiles. Only an average of one node of Ranvier per 24 myelinated profiles was observed. There was a wide range (1-14) of nodes per specimen. Scatter diagrams for number of neurotubules and neurofilaments versus axon circumference are illustrated in Figs. 7 and 8, respectively. The method of least squares was used to fit polynomial curves to the data. The best curves and their equations are displayed on the scatter diagrams. The size distribution of unmyelinated profiles fully invested by Schwann cell cytoplasm is compared to the distributions of partially and completely non-invested unmyelinated profiles in Fig. 9. Of the 2083 unmyelinated profiles analysed, about 50-
. .
45T = (16.8
40-
ym-1)
C -
6.3 .
r = 0.786 . .
35-
. l.
30-
. /
c
. .
l
.
g
. .
. .
.
IO-
.
l
5-
.:
.
.
. l
.
l
. *
*as 8 l . . . ..
l e8 . . . i//-/
o0
.
.
. .
.
.
a .
.
.
20
I.0 Circumference
(C)
in
ym
FIG. 7. Scatter diagram for number of neurotubules per nerve profile versus the axon cross-section circumference. The method of least squares yields the straight line as the best fit to the data.
QUANTITATIVEORGANLZATIONOFINTRADENTALNERVEINMARMOSET
651
300-
. 250_
F = (39.6 p&) C* - (II.2
pm-l)
C + 73
. l
.
2 ,200z E 0 = + 0
150-
L _z 5 z
IOO-
50-
20
I.0 Circumference
in
urn
FIG.8. Scatter diagrams for number of neurofilaments per nerve profile vs. the axon cross-section circumference. The method of least squares yields the parabola as the best fit to the data.
30 per cent were only partially invested by Schwann cells.
invested
while about
4 per cent were completely
non-
DISCUSSION
The subdivision of fibres into “core” and “periphery” was only based on the definition of the group of fibres constituting the core (Fig. 1). The basis for subdivision was therefore arbitrary but served the interpretative purpose of distinguishing between fibres entering the tooth and destined to more coronal portions of the pulp (core fibres) from those following the vasculature or those preterminal fibres innervating the odontoblasts at or near that cross-section. More unmyelinated than myelinated nerve fibres were consistently observed within the intradental nerve cores. This large amount of unmyelinated nerve fibres entering the tooth must undoubtedly have a major contribution to the physiological processes within it. These results are in conflict with previous publications that report fewer unmyelinated fibres (WINDLE, 1927; GRAF and BJBRLIN, 1951; UCHIZONO and HOMMA, 1959; PISCHINGER and STOCKINGER, 1968).The difference is due to the inability of the light
652
K. W. BUELTMANN,U. L. KARLSSONAND J.
EDIE Number
.
.
Completely
89
non-invested
._______, Partially
596
invested +.
_.
-.
Completely
2 Circumference I 0.5 Equivalent
1398
in
Am
I I.0 diameter
in
yrn
FIG. 9. Fibre circumference distributions of fully invested (solid and dashed line) partially invested (dashed line) and non-invested (solid) nerve profiles. The greater the degree of
investment, the more likely the nerve profile will assume large circumferences. The class interval width was 0.27 pm. microscopic and previous histologic techniques to observe adequately and count the unmyelinated fibres. In this investigation, all the observed myelinated fibres were counted and measured. Very few escaped detection as only parts of the odontoblastic layer was missing from each specimen. The number of nerve fibres per tooth is comparable to that obtained from human teeth by GRAF and BJ~~RLIN (1951). With all the in-vitro criteria for nervous tissue preservation fulfilled (CAULFIELD, 1957; ROBERTSON, 1960; SJ~~STRAND,1967; BERTHOLD, 1968), both the unmyelinated and, in particular, the myelinated fibres were found to have irregular, rather than circular, contours. BERTHOLD(1968) suggested that this irregularity in form is a direct reflection of their in-vivo shape which results from the myelin sheath adapting its shape to localized
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accumulations of the surrounding Schwann cell cytoplasm. If this view is acceptable, the circular profiles resulting from previous preservation methods must be deemed artifactual. A comparison of previous diameter measurements with the circumference measurements of this investigation is possible if it is assumed that the axon circumference retains the same length for both electron and light microscopic preservation methods. Then, the comparable diameters (equivalent axon diameters, C/n, plus twice the average myelin sheath widths) from this investigation are, on the average, 0.5 pm larger than those found by GRAF and BJ~RLIN(1951). There was no apparent difference in the size of the myelinated fibres in the core and periphery (Fig. 6). The increased measurement precision and relative shape independence using electron microscopic techniques should permit more reliable results. However, the validity of any geometrical measurement must await the establishment of the true in-vivo shape of nerve fibres. The unmyelinated fibre-size distribution (Fig. 6) conflicts with that reported by UCHIZONOand HOMMA(1959). Their unimodal diameter distribution had a range from 0 *5 to 2 *5 pm, a modal value of about 1 *Opm and an overlap with the myelinated spectra from 1 .O to 2 *5 I_cm(including 45 per cent of all fibres). The present study shows a much smaller overlap (less than 5 per cent), a range of circumference measurements from 0.8 to 1.6 pm (equivalent diameters from 0.25 to 0 *50 pm) and a modal value of about 1 .O pm (0.3 pm equivalent diameter). It is unlikely that these discrepancies can be attributed to species differences and different processing methods alone. The cross-sectioned core exhibited all the ultrastructural characteristics of a peripheral nerve. Only the lack of fasciculation and a nerve sheath were observed. This may be reflected functionally in an increased sensitivity of the nerve to mechanical stimuli. Indeed, pulsating electrophysiologic responses, as originating from pulpal vessels, have been recorded (FUNAKOSHIand ZOTTERMAN,1963). One may even question the nerve ending per se as being the only location for stimulus since naked nerve fibres, such as those observed here (Fig. 4), may be very sensitive to environmental changes. If true, the functional concept of the nerve ending may then be extended to the preterminal part of the intradental nerve. This view would have to be reconciled with those discussed in recent publications (EIFINGER,1969; S~OCKINGER and PRITZ, 1970). The presence of non-invested nerve fibres does not necessarily imply that unmyelinated fibres are non-invested along their entire course since this was a two-dimensional analysis. They may actually represent “nodes” of unmyelinated fibres since they are thinner than the invested profiles (Fig. 9). An apparent corroboration exists in that nodes of the myelinated fibres appear with the same relative frequency in our material. This interpretation raises a question about the exact mode of the propagation mechanism in unmyelinated nerves. The data correlating neurofilament and neurotubule numbers to axon circumference and area, respectively, appears to be a quantitative and geometrical account of observations noted by PISCHINGERand STOCKINGER,(1968). They noted that the smaller axon profiles contained mostly filaments.
654
K. W. BUELTMANN,U. L. KARLSSONANDJ. EDIE
The apparent proportionality between the number of neurotubules and axon circumference suggests the neurotubules are arranged in curvilinear patterns. It appeared that the main pattern was a circle concentric with the axon axis but with some tubules surrounding larger organelles. There were, on the average, 17 additional tubules for each micrometer circumference increment above 0.4 pm. On the other hand, the parabolic relationship between the number of neurofilaments and axon circumference suggests the neurofilament number is dependent upon axon cross-sectional area and that the neurofilaments are distributed with uniform density within the axon. An examination of the micrographs reveals that the distribution excludes the periphery of the axon’s interior. There were, on the average, 40 additional filaments for every additional square micrometer of axon cross-sectional area. Acknowledgements-Supported by intramural funds from the Graduate College, Department of Periodontology, Dental Research Laboratory, from the NIH General Research Support Grant award to College of Dentistry and from the Neurosensory Center, Program Project Grant Number NSO 3354 of the National Institute of Neurological Diseases and Stroke. R&umb-On a investiguC quantativement le nombre, les circonfbrences des axons, les quantites de mykline, les couvertures desnerfs et l’organisation intra-axionale, dans des sections transversales apicales de la pulpe de la dent chez les marmottes, pour les fibres nerveuses mytlinCes et non-mytlinCes. Les pulpes ont 6tB preservees in situ par perfusion d’aldkhyde et prtparkes pour la microscopic Clectronique. Le nombre des fibres nonmytlinCes dCpassait les fibres my6lin6es, la difft%ence dans les distributions de leurs dimensions ttant moindre que 5 pour cent. Des nombreuses fibres non-myClinCes Ctaient partiellement ou totalement non-couvertes et cons&quemment d’une dimension plus rCduite. On a remarque des neurotubules et des neurofilaments ayant des configurations gComttriques particuli&es, dependant de leur dimension. Ces rtsultats devraient aider ?I Ctablir une base ultrastructurale quantitative pour l’organisation de la fibre nerveuse B I’int&ieur de la pulpe de la dent. Zusammenfassung-Es wurden eine Anzahl von Neuritenumfgngen, Myelinbreiten, Nervenumhiillungen und interaxonaler Organismus mengenmgssig fiir myelinisierte und nicht myelinisierte Nervenfasern in Apikalquerschnitten der Pulpa des ausgewachsenen Krallenaffen untersucht. Die Pulpas wurden in der natiirichen Lage durch Ubergiessen mit Aldehyd prgserviert und ffir Elektromikroskopie behandelt. Die Zahlen der nicht myelinisierten Fasern iiberschritten die myelinisierten Nervenfasern und die Umfangsverzweigungen iiberlappten in Grijsse urn weniger als 5 Prozent. Viele, nicht myelinisierte Fasern waren mit entsprechender kleineren Massen teilweise oder vdllig nicht umhiillt. NeurokanPlchen und Fiserchen besassen nach der Beobachtung besondere und grtissenabhlngige, geometrische Bildungen. Diese Resultate sollten dazu beitragen, eine mengenmgssige, ultrastrukturelle Grundlage fiir den Nervfaserorganismus innerhalb der Zahnpulpa festzulegen. REFERENCES BERTHOLD,C. H. 1968. Ultrastructure of the node-paranode region of the mature feline ventral lumbar spinal-root fibres. Acta Sot. Med. Upsal. 73,37-67. CAULFIELD,J. B. 1957. Effects of varying the vehicle for 0~0, in tissue fixation. J. Biophysic. Biochem. Cytol. 3, 827-830.
DELANGE, A., HANNAM,A. G. and MATTHEWS,B. 1969. The diameters and conduction velocities of fibres in the terminal branches of the inferior alveolar nerve. Archs oral Biol. 14,513-519.
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EIFINGER, F. 1969. Zum Problem der Pulpa-Dentin-Innervation. Dt. Zahn-, Mund- Kieferheilk., B6 58, H 5/S, 188-201. ENGSTR~M,H. and &MAN, A. 1960. Studies on the innervation of human teeth. J. dent. Res. 39, 799-809. FRANK, R. M. 1966. Ultrastructure of human dentine. In: Third Europ. Symp. on Calcified Tissues (edited by FLEISH,BLACKWOODand OWEN), pp. 259-272. Springer, Berlin. FRIEDE, R. L. and SAMORAJSKI, T. 1967. Relation between the number of myelin lamellae and axon circumference in fibers of vagus and sciatic nerves of mice. J. camp. Neurol. 130-131,223-231. FUNAKOSHI,M. and ZOTTERMAN, Y. 1963. A study in the excitation of dental pulp nerve fibres. In: Sensory Mechanisms in Dentine. (Edited by ANDERSON,D. J.) pp. 60-72. Pergamon Press, Oxford. GANONG, W. J. 1967. In: Review of Medical Physiology, p. 32. Lange Medical Publications, Los Altos. GRAF, W. and BJORLIN,G. 1951. Diameters of nerve fibers in human tooth pulps. J. Am. dent. Ass. 43,186193.
GRAF, W. and HJELMQUIST,U. 1955. Caliber spectra of dental nerves in dogs and cattle. J. camp. Neurol. 103,345-353. GREGORY,W. K. 1922. The Origin and Evolution of the Human Dentition, pp. 218-219, Baltimore, Maryland. KARLSSON,U. and SCHULTZ,R. 1965. Fixation of the central nervous system for electron microscopy by aldehyde perfusion. J. Ultrastruct. Res. 12, 160-186. MATTHEWS,B. 1968. Cold-sensitive and heat-sensitive nerves in teeth. J. dent. Res. 49, 974-975 (Abstract). MATTHEWS,J. L., DORMAN,H. L. and BISHOP,J. G. 1959. Fine structure of the dental pulp. J. dent. Res. 38, 940-946. NEIDLE,E. A. and LIEBMAN,F. M. 1964. Effects of vasoactive drugs and nerve stimulation on blood flow in the tooth pulp and allied structures of the cat. J. dent. Res. 45,412422. PISCHINGER,A. and STOCKINGER, L. 1968. Die Nerven der menschlichen Zahnpulpa. Z. Zelfjbrsch. 89, 44-61. ROBERTSON,J. D. 1960. The molecular structure and contact relationships of cell membranes. Prog. Biophys. 10, 343418. SCOTT,D. JR. and TEMPEL,T. 1963. Receptor potentials in response to thermal and other excitation. In: Sensory Mechanisms in Denrine (Edited by ANDERSON,D. J.). Oxford, Pergamon Press. SJ~STRAND,F. S. 1967. Electron Microscopy of Cells and Tissues, Vol. l., p. 410, Academic Press, New York. STOCKINGER,L. and Pturz, W. 1970. Morphologische Aspekte der Schmerzempfindung im Zahn. Dr. Zahn. Z. 25(5), 557-565. UCHIZONO,K. and HOMMA,K. 1959. Electron microscopic studies on nerves of human tooth pulp. J. dent. Res. 38, 1133-l 151. WINDLE, W. F. 1927. The distribution and probable significance of unmyelinated nerve fibres in the trigeminal nerve of the cat. J. camp. Neurol. 41,453-477.
PLATES1-5
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PLATE 1 FIG. 1. A cross-section of dental pulp close to the apex of a marmoset incisor tooth. The rectangular outline encloses the bundle of nerve fibres referred to as “core “fibres. Those fibres beyond this outline and with no obvious connection to the core fibres constitute the “periphery”. O-5 pm section, methylene blue. x 80
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PLATE 2
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PLATE 2 FIG. 2. Survey electron micrograph of the intradental nerve core that is preserved by glutaraldehyde/OsO, fixation. Note the irregular contours of the myelinated profiles, the variation of myelin sheath thickness and the groups of unmyelinated profiles (U). It was necessary to examine the unmyelinated profiles at higher magnifications before their true identities could be established. In the lower half. a fibroblast process @) is traversing the core. A nucleated Schwann cell (S) and a profile sectioned close to a node of Ranvier (R) can be seen. x 10,300
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PLATE 3 FIG. 3. A group of unmyelinated profiles are seen surrounded by variable amounts of a Schwann cell process. The Schwann cell cytoplasm (S) is more opaque than the
axoplasm, which displays a typical organization of neurotubuIes (T) at the periphery and neurofilaments (F) in the interior of the larger profiles. The nerves are separate from the investing Schwann cell cytoplasm by a SO-200 A space. x 72,000
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PLATE 4 FIG. 4. A non-invested nerve profile is displayed below. It is not invested by Schwann cell membrane, but is surrounded by basement membrane material (BM). The profile is identified as a nerve process since it exhibits the characteristic neurotubule/neurofilament organization. Above is seen part of a myelin sheath with surrounding Schwann cell invested by basement membrane material (BM). x 184,000
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PLATE 5
FIG. 5. A myelinated profile is seen sectioned in its para-nodal region. Note the diikrence in the opacities of the Schwann cell cytoplasm (S) and the axoplasm (A). The neurotubules (T) and vesicles (V) are arranged along the periphery of the nerve process and around mitochondria (M). Filaments (F) cover the remaining surface. The opaque granular material in the Schwann cytoplasm was typically found in the para-nodal regions of the fibre. (About x 100,000)
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IN MARMOSET
PLA .TE 5
A.O.B.
f.p. 660