Electron microscopic quantitations of feline primary and permanent incisor innervation

ELECTRON PRIMARY

MICROSCOPIC QUANTITATIONS OF FELINE AND PERMANENT. INCISOR INNERVATION D. C. JOHNSENand U. L. KARLS~ON

Ultrastructure Laboratory and Departments of Anatomy and Pedodontics, Colleges of Dentistry and Medicine, University of Iowa, Iowa City, Iowa 52242, U.S.A. Summary-Axons from pulps of fully developed primary and permanent teeth were processed for electron microscopy, counted and measured for cross-sectional area and circumference. These measurements were totalled for each specimen and experimental groups were compared statistically. Permanent teeth contained a significantly greater myelinated and unmyelinated innervation However, primary teeth contained a higher proportion of unmyelinated axons. Considering the tooth weight as an indication of the amount of tissue innervated, no significant differences were found in amount of innervation between primary and permanent teeth. Therefore, innervation density appears the same, while relative proportions of fibre types are not. Fibre-size analyses suggested that myelinated axons in permanent tooth pulps were more irregular in shape. It is concluded that differences exist between the innervation patterns of primary and permanent teeth. These differences may be relevant to tooth sensitivity. A significant positive linear correlation was found between amounts of myelinated and unmyelinated innervation in primary teeth. This suggests predictability of unmyelinated innervation quantities from more easily determined myelinated innervation quantities.

INTRODUCTION Reception of an external stimulus by the tooth is the first phase of a neurological sequence for passage of an impulse to the higher nerve centres with perception as the result. It has been suggested that the innervation pattern of primary teeth is similar to permanent teeth (Rapp, Avery and Strachan, 1967). However, clinical observations support the notion that children react differently from adults to dental stimuli. The innervation of the primary tooth has never been quantitated, nor has the innervation of primary and permanent teeth been adequately compared. The trigeminal nerve has been shown to be the major source of innervation for teeth (Windle, 1927). Sympathetic axons have been inferred to account for only a small part of pulpal innervation (Ogilvie, 1969). The presence of parasympathetics is still uncertain. Nerve branching is most frequent in the coronal portion of the pulp with very little branching in the root portion (Engstrom and Ohman, 1960). The dental pulp contains both myelinated and unmyelinated nerve fibres (Matthews, Dorman and Bishop, 1959). The presence of myelinated axons in the range of 1 pm5 pm in diameter suggests a sensory role (Harris and Griffin, 1968). Electron microscopic studies have shown that cross-sections of myelinated nerve fibres display irregular profiles (Miyoshi, Nishijima and Imanishi. 1966). Unmyelinated axons dominate in number and are usually less than 1 pm in diameter (Bueltmann, Karlsson and Edie, 1972). Pulpal nerves appear to be structurally similar to other peripheral sensory nerves.

Only a few studies have ascertained the number of axons entering the permanent tooth. With the light microscope, the number of entering myelinated fibres has been demonstrated for humans (Graf and Bjorlin, 1951) and experimental animals (Graf and Hjelmquist, 1955). Numerical determinations of unmyelinated axons has been adequately determined with the electron microscope only in marmosets (Bueltmann et al., 1972). Assuming that velocity and amplitude of nerve impulses are related to fibre size (Erlanger and Gasser, 1937), intradental axon dimensions just within the apex should approximately correlate to the total impulse flow which accumulates from the entire tooth. The primary objectives of this study were to determine some dimensional cross-sectional parameters of intradental axons from a suitable number of primary incisor pulps in the cat and to compare these findings with permanent tooth pulps.

MATERIALS AND METHODS Nine fully developed mandibular primary central incisors from six domestic cats (Fe/is dornesticcr) aged 58 weeks and three mandibular permanent central incisors from two adult cats aged 2 yr were selected for the study. Surgical anaesthesia was obtained by 25 mg sodium pentobarbital (Nembutal, Abbott) intraperitoneally (i.p.) per kilogram body weight. The animals also received 10,000 USP units i.p. of isotonic aqueous sodium heparin. The fixation procedure using glutar671

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aldehyde perfusion was essentially that described by Karlsson and Schultz (1965). Following perfusion. an antereposterior radiograph was taken of the anterior mandible in order to ascertain the stage of development and the lack of pathological processes. The central incisor teeth were dissected out and examined for evidence of resorption. Connective tissue was scraped from the roots and the teeth were weighed within 1 min. The hard tissue of the tooth served as a capsule for the pulp, preventing dehydration. Tooth weight was used as an indication of the amount of tissue innervated by intradental nerves. Apical portions of the pulps were removed by carcfully cracking the teeth in a small vice and dissecting away the fragments. Pulpal tissue was rinsed in isotonic phosphate buffer and postfixed in 1 per cen.t buffered osmium tetroxide for 2--4 hr. After a second buffer rinse, specimens were dehydrated in increasing concentrations of acetone and embedded in Vestopal W. Cross sections from the juxta-apical specimens of the pulps were cut at 7@~100 pm for electron microscopy. Sections were collected on Formvar films supported by single-hole grids and stained first at 60°C with a saturated solution of uranyl acetate for 30 min and then for I min with lead citrate.

The specimens were observed and photographed in a Siemens 101 electron microscope. Photography. counting and circumference measurements of myelinated and unmyelinated axons were accomplished bq a technique described by Bueltmdnn c’t (11. (1972). Axonal cross-sectional areas were measured with ;I compensating polar planimeter (KeulTel and Esser Company) which had a measurement error of 2 per cent. The following definitions were used: Innervation valuesconsist of the number, total crosssectional area and total circumference of myelinated, unmyelinated and total axons. Innervation indices relate the innervation values to the weights of the respective teeth and were derived by dividing each innervation value by the tooth weight. The core constituted the central area of the pulp, containing the majority of the myelinated fibres. The periphery constituted the remainder of the pulp.

RESL!LTS

Eleven of the 12 specimens demonstrated adequate membrane and organelle integrity. One specimen. a permanent tooth pulp, demonstrated marginally acceptable fixation. It was used only for determination of

Table 1. Statistical analysis of innervation values and ratios for entire pulps of nine fully developed primary teeth and 2 fully developed permanent teeth. Three fully developed permanent teeth were used for tooth weight and myelinated number. The following abbreviations are used in the table and in Fig. I. Wt.: weight in mg.; My.: myelinated axon; Umy.: unmyelinated axon; X: myelinated plus unmyelinated axons; No.: number; Area: total cross-sectional area of one type(s) of axon in /lrn’; Cir.: total cross-sectional circumference for one type of axon in kern Tooth wt.

MY. no.

MY. area

MY. cir.

Umy. no.

Umy. area

Umy. cir.

2.14 0.50

22 15.9

39.13 26.64

122.20 81.00

149 109.94

15.21 9.68

167.07 1 IO.88

157.96 55.65 4.87’r

769.90 106.29 9.84t

432 62.93 3.42t

27.67 0.47 1.75

40 1.46 30.43 2.85*

Primary pulps Mean S.D. Permanent Mean SD.

pulps 6.95 0.79 1I .49t

126 30.9 7,81-I,

C No.

C area

1 cir.

My. no.: umy. no.

My. area: umy. area

My. cir.: umy. cir.

Primary pulps Mean 171 S.D. 122.92

54.35 34.27

288.13 181.24

0.14 0.07

2.54 I.35

0.7 I 0.33

185.63 56.12 4.5O.t

1171.20 137.04 6.397

034 0.05 3.60t

5.70 I .92 2.831

I .92 0.12 4.95$

t

Permanent Mean S.D.

t

pulps 575 60.8 I 4.39t

* p < 0.05. t p < 0.01.

Quantitations of primary and permanent teeth innervation

B

MI/WI

M~.Arao/Wf

MyCir/Wl.

Un

‘Wt. UmyArea/Wf

“myC,r,W+

17Wl

ZArea/Wt.

673

EClr/Wt.

Fig. 3. Graph showing quantitative relationships of innervation indices of fully developed primary compared to permanent teeth. The mean for each innervation index is adjusted graphically so that permanent teeth can be used as a reference. Values for primary teeth are graphically adjusted accordingly. Note that for primary teeth myelinated indices are lower, unmyelinated indices are higher and total indices are similar. See Table 1 for explanation of abbreviations.

myelinated axon number since myelin sheaths were intact. All specimens contained centrally located group(s) of myelinated axons surrounded by connective tissue. These characteristics indicated the completeness of the dissections. No general morphological or statistically significant quantitative differences were found between pulps from unresorbed primary teeth and those with evidcncc of beginning resorption. Pulps from primary teeth all had one or two groups of myelinated axons located near the centre of each cross section (Fig. 1). This core of myelinated axons occupied only a small part ofthe total cross-sectional area of the pulp. In the much larger peripheral area, myelinated axons were rarely present. Pulps from permanent teeth had many more myelinated axons than primary pulps (Table 1) and the core occupied a large proportion of each cross section (Fig. 2). Innervation values were higher for permanent teeth as shown in Table 1. The mean number of myelinated axons for primary teeth was 22 (range l-43) and for permanent teeth 126 (9&142). The differences between permanent teeth and primary teeth were the greatest for myelinated innervation values (my. no., my. area and my. cir.). All of these differences were statistically significant. Total innervation values (X no., C area and X cir.) also showed a statistically significant difference between permanent and primary teeth. However, unmyelinated innervation values showed less difference between the two groups. The number of unmyelinated axons (umy. no.) and cumulative circumference of unmyelinated axons (umy. cir.) were significantly higher for permanent teeth. No significant difference was found for cumulative unmyelinated axons (umy. area) for the two types of teeth. mary teeth were high.

Standard

deviations

for pri-

Innervation indices reflected no statistically significant difference between permanent and primary teeth (Fig. 3). Naturally, this was related to the fact that permanent teeth were heavier. The unmyelinated innervation indices were somewhat higher for primary teeth. The total innervation indices were very close for permanent and primary teeth.

ml

My

Umy.

Perm.

Primary

look

00%

60%

40%

20%

-

-

#

Perm.

Primary

Area

Penn

Pri

mory

Cir

Fig. 4. Graph showing percentages of innervation values of myelinated and unmyelinated axons for fully developed primary and permanent teeth. Uniform dominance for permanent teeth is evident.

D. C. Johnsen

674

and U. L. Karlsson -------Permanent -Primary

LL

0

.6

I2 I.824

_-_-__.a

30 3.6 4.2 4.6 54 Myelinated

Fig. 5. Graph

showing

Nerve

GO 66

72

76

Circumference

84 90

96

IO.2 108 114 120 130

in,Um

size spectra for myelinated nerve fibre circumference in fully developed and permanent cat incisors. Class intervals are @6 urn.

The predominance of myelinated or unmyelinated innervation differed depending on the innervation values examined, as shown in Fig. 4. The number of myelinated axons comprised a small percentage of the total number of axons. However, the cross-sectional area of myelinated axons made up well over half of the total cross-sectional area of all the axons. Total axon circumference was more evenly divided between myelinated and unmyelinated axons. The ratios of myelinated to unmyelinated innervation values differed for primary and permanent teeth as seen in Table I and Fig. 4. Myelinated innervation ratios were significantly higher in permanent teeth. The distributions ofmyelinated axon circumferences were unimodal for primary and permanent tooth pulps and the curves were almost identical. Peaks for both curves were between 4.80 and 539 pm. Little skewing was observed (Fig. 5). The distributions ofmyelinated axon areas were also unimodal in both primary and permanent tooth pulps. The peaks for myelinated axons were between 0.80 and I:19 pm2 for primary pulps and between 060 and 0.79pm’ for permanent pulps. There was a trend for permanent teeth to have relatively smaller axons. Consequently. the curve for permanent teeth was more skewed to the left than the one of primary teeth (Fig. 6).The distributions of unmyelinated axon areas presented unimodal skewed distributions for primary and permanent teeth. All curves were skewed to the left and all had peaks between 0.1 and 0.4 pm2. Curves for permanent tooth cores and peripheries and for primary tooth peripheries were similar. The curve for primary tooth cores showed a slight tendency toward larger axons with respect to cross-sectional area. Less than 5 per cent overlap was evident between curves for myelinated axon area and unmyelinated axon area. Unimodal. skewed distributions were found for unmyelinated axon circumferences. Peaks for all four curves fell between 0.60 and OX9 pm. The curve for pri-

primary

------.Permo”e”t -l%mary

,201

Fig. 6. Graph showing size spectra for myelinated axon area in fully developed primary and permanent cat incisors. Class intervals are 0.2 pm2 up to 4.0 /cm?.

4’ i

Fig. 7. Scatter diagram showing relationship of myelinated axon number to unmyelinated axon number in fully devcloped cat primary incisors. Each point represents one pulp.

Quantitations of primary and permanent teeth innervation mary tooth cores showed a very slight tendency toward larger axons. Less than 5 per cent overlap was found between curves for myelinated and unmyelinated axon area. A statistically significant, positive, linear relationship existed between myelinated and unmyelinated innervation values in fully developed primary teeth. Correlation coefficients for myelinated versus unmyelinated innervation values were as follows: myelinated to unmyelinated axon number, r = 0.788 (p < 0.05), myelinated to unmyelinated axon area, Y = 0.725 (p < 0.05) and myelinated to unmyelinated axon circumference. r = 0.823 (p < 0.01). As an example, the regression of myelinated axon number against unmyelinated axon number is presented graphically in Fig. 7. DISCUSSlOiV

The overall appearance of cat permanent incisor pulps in this study is similar to marmosets and humans as reported previously (Bueltmann et al., 1972; Graf and Bjorlin, 1951). The large number of myelinated axons located centrally in the pulp is considered reason enough to accept these three specimens as representative samples of cat permanent incisor pulps. Generally, conduction velocities and spike sizes are related to axon diameters as revealed by light microscopic techniques (Erlanger and Gasser, 1937). However, it is well known that axon volume is affected by processing procedures for both light and electron microscopy. Therefore, both axon circumference and area parameters were obtained in this investigation. The rationale was that the combination would better represent the dimensional characteristics than either single parameter. The combination will presumably be of more value for future efforts to correlate dimensions and physiological characteristics. In the present study, emphasis is not placed on the classical function-anatomic dimensions relationship --due to the uncertainty in determination of nerve diameter. However, for purposes of universality, categorization of results is mentioned in traditional terms. The categorization is made by conversion of circumferences and area found in the present study to diameters. Myelinated axons appear to fall into the A-delta category suggesting pain conduction. Unmyelinated axons appear to fall into the C-fibre category associated with pain conduction and autonomic function. Only a small part of the axons may be assumed to be autonomic (Ogilvie, 1969). It is possible that autonomic innervation accounts for differences in relative abundance of axon types in primary and permanent teeth. Although unmyelinated indices of primary pulps appear to be slightly larger than for permanent pulps, the similar abundance of blood vessels would suggest a similar amount of autonomic innervation for both types of teeth. If so, the autonomic part of the nerve population would be constant and the major part mainly concerned with dentine sensitivity. Consequently. this discussion concerns mainly the

675

tooth as a pain receptor since other modes of sensation appear not to have been decisively documented. Significantly different innervation values for pulps from primary and permanent teeth suggest a higher absolute capability for impulse transmission in permanent teeth. However, the overall appearance of the permanent pulp filled with nerves may be somewhat misleading compared to the sparsely populated primary ptilp. An indication of amount of tissue innervated must also be considered, i.e. the relationship to the tooth weight. The observed similarity in innervation indices for primary and permanent tooth pulps would not lend support for a hypothesis of differences in nerve impulse transmission capability between the two types of teeth, considering tooth size. However, the observed significant differences in abundance of myelinated vs unmyelinated axons between primary and permanent teeth may be of importance. Known physiological differences include higher conduction velocities, greater spike intensity (Erlanger and Gasser, 1937) and lower impulse threshold (Brazier, 1968) in myelinated axons. It has also been suggested that small diameter, sensory, myelinated axons transmit a sharp shooting pain while unmyelinated axons transmit a dull lingering pain (Bigelow et al., 1945). In terms of this investigation, one suggestion would be that permanent teeth appear to have a greater potential for conduction of sharp pain. It has been shown that unmyelinated axons are more easily anaesthetized with procaine than myelinated axons (Sinclair, 1967). Although an overlap has been demonstrated, the relative differences as shown here may be significant with respect to anaesthetic susceptibility of primary compared to permanent teeth. We have no obvious explanation for the higher linear correlation of myelinated and unmyelinated innervation values in primary teeth. It suggests that unmyelinated innervation values can be predicted if myelinated values are known. The significance is that a myelinated axon count from a light micrograph may be sufficient for estimation of average total innervation. It is unknown whether axons in the pulp periphery are branches of more proximal core axons or if they represent separate axons entering the pulp. Interestingly enough, there seems to be neither a detectable difference in individual sizes between core and periphery nor in the number of axons enclosed by a single Schwann cell in the same experimental group. From qualitative observations and from axon size distributions, it appears that a different general shape exists for myelinated nerves in the two groups of specimens. Similarity in circumference curves but a difference in area curves shows that primary teeth tend to have myelinated axons that are similar in circumference, but larger in cross-sectional area. Cross-sectional shapes which would fit these findings are an axon with an irregular outline for permanent teeth and a more rounded axon for primary teeth. Reasons for the difference in myelinated axon shape may lie with different

676

D. c‘. Johnsen

and U. L. Karlsson

reactions to the tissue processing or with the age differential of the two groups of teeth. A decrease in nerve diameter with age has been suggested (Sinclair, 1967) but such determinations are arbitrary for irregularly shaped nerves. The use of circumference and area avoids this difficulty. Assuming age to be the explanatory factor. it is suggested that the myelinated axons maintain their circumference while undergoing a loss in axoplasm volume in the ageing process. Functional capabilities for the two shapes of nerves are unknown, but may differ. In conclusion, some characteristic nerve features appear to be similar in primary and permanent cat teeth. e.g. innervation indices and myelinated axon circumferences. However. there are also several differences. As compared to permanent teeth, primary teeth contain (1) significantly fewer axons, but relativeI> more and larger unmyelinated axons and (2) myelinated axons that te;ld to have a more rounded shape. Although other pulpal differences have been observed here and/or are known from other investigations, these data generally suggest that peripheral nerve characteristics should be taken into account when physiological and psychological diflerences of pain perception are evaluated. .4CkrloM.fudgP/,tu,IT\~~This investigation was supported by the PHS General Research Support Grant to University 01

Iowa. Colleges of Dentistry and Medicine. and the Neurosensory Center. Program Project NSO- 3354 National Institute of Neurological Diseases and \;euroacnsor\ (‘cnrcr I’uhl~cat~on humhcr ;22.

of the Stroke.

Bigclow h.. Harrlaon I.. Goode11 H. and Woltr H. G. 19-15. Studies on pain. quantitative measurements of two pain

sensations on skm, with reference to the nature of hyperalgesia of peripheral neuritis. J. clin. Inaest. 24, 503-5 12. Brazier M. A. B. 1968. Thr Electrical Acticity of rhe Nrruous Sltrr,r. Third edn. pp. 19~42. Williams & Wilkins Co.. Baltimore. Bueltmann K. W.. Karlsson U. L. and Edie J. 1972. Quantitative ultrastructure of intradental nerve fibres in marmosets. ,4rc/ls oral Biol. 17, 645-660.

Engstrom H. and ijhman A. 1960. Studies on the innervation of human teeth. .I. dent. Res. 39, 799.-809. Erlanger J. and Gasser H. 1937. Electricul Signs c?f Nercou.~ Actiuily. University of Pennsylvania Press. Graf W. and Btorlin G. 1951. Diameters of nerve fibres in human tooth pulps. Am. ht. J. 43, 186 193. Graf W. and Hielmauist V. 1955. Caliber soectra of denldl nerves on dogs anh cattle. J. cornp. Nrurk 103. 345- 353. Harris R. and Griffin C. J. 1968. Fine structure of nerve endings in the human dental pulp. Irchs. wctl Rio/. 13, 773 77x. Karlsson U. and Schultz R. L. 1965. Fixation of the central nervous system for electron microscopy by aldehyde perfusion. J. Cltrastruct. Rex 12, 16&186. Matthews J. L.. Dorman H. L. and Bishop J. G. 1959. Fine structure of the dental pulp. J. dent. Rrs. 38, 94&946. Miyoshi S., Nishijima S. and Imanishi I. 1966. Electron microscopy of myelinated and unmqelinated nerve fibrcs in human pulp. Archs orul Biol. 11, 845-846. Ogilvie R. W. 1969. The vasomotor innervation of the cats’ lower right canine tooth pulp. .4mt. Rec. 163, 347. Rapp R.. Avery J. K. and Strachan D. S. 1967. The distribution of nerves in human primary teeth. 4rzat.Rec. 159,8993. Sinclair D. 1967. C‘utuwousSrmsuriou, pp. 5% 80. IO1 IZI and 139 155. Oxford University Press. London. Wlndlc W. F. 1927. Experimental prdof of the types of neurons that innervate the tooth pulp. J. camp. Nwrol. 43. 337 356.

l&urn& Les axones de pulpes de dents temporaires et permanentes ad&es sont Ctudiks en microscopie Clectronique. dttnombris et mesurks en diametre et en circonftrence. Ces mesures sont totaliskes pour chaque specimen et les groupes expkrimentaux sont cornpar& statistiquement. Les dents permanentes prCsentent une innervation myClinisCe et amyClinisCe nettement plus importante. Ccpcndant les dents permancntcs prisentent une proportion plus klev&e d’axones amy~linis&. En tenant compte du poids de la dent, comme indication de l’ensemble du tissu inner& aucune diffirence significative cn quantitL; d’innervation entre les dents temporaires et permanentes n’est notCe. Par suite. la densite d’innervation semble identique, alors que les proportions relatives de tvpes tibrillaires sont difikrentes. Une analyse de la taille des fibres montre que les axones my&Ii&s des pulpes de dents permanentes sont de taille plus rCguli&re. Des diff&rences de types d’innervation des dents temporaries et permanentes existent done. Ces diffkrences peuvent &re importantes pour la sensibilitk de la dent. Une corrClation IinCaire positive et significative est observCe entre les proportions d’innervation myClinisCe et amyttliniske dans les dents temporaires. La proportion d’innervation amyilinide peut done Ctre dCduite en dCterminant I’inncrvation myt-linisie plus facilement dknombrke.

Zusammenfassung-- Die Axone aus vollstiindig entwickelten Pulpen von Milch- und bleibenden Zlhnen wurden zur elektronenmikroskopischen Untersuchung vorbereitet, gezBhlt und im Durchmesser und Umfang ausgemessen. Diese Bestimmungen wurden fiir jeden Einzelfall 7usammengestellt und in Versuchsgruppen statistisch verglichen. Bleibende Z%hne zeichneten such durch tine signifikant st&rkere Innervation mit markhaitigen und marklosen Fasern aus.

Quantitations of primary and permanent teeth innervation MilchzPhne enthielten dagegen einen hiiheren Anteil markloser Axone. Mit Bezug auf das Zahngewicht als MaD ftir das innervierte Gewebe lieBen sich zwischen Milch und bleibenden Ziihnen keine signifikanten Differenzen fiir das AusmaB der Innervation finden. Daher scheint die Innervationsdichte gleich zu sein, wlhrend dies ftir die relativen Anteile der Nervenarten nicht zutrifft. Analysen des Faserumfanges deuten darauf hin, da13 markhaltige Axone in den Pulpen bleibender Zlhne von unterschiedlichem Umfange waren. Daraus wird geschlossen, da0 im Gesamtbild der Innervation zwischen Milch- und bleibenden Zlhnen Unterschiede vorhanden sind. Diese Unterschiede miigen fiir die SensitivitHt der Ziihne verantwortlich sein. Eine signifikant positive lineare Korrelation wurde zwischen den Anteilen markhaltiger und markloser lnnervation in Milchzlhnen gefunden. Dies 1lBt es zu, die Anzahl markloser Fasern aus der leichter feststellbaren Quantitgt markhaltiger Nervenfasern vorauszusagen.

PLATE I OVERLEAF

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D. C. Johnsen

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and U. L. Karlsson

Fig. 1. Electron micrograph of a juxtaaapical pulpal cross section from a fully developed cat primary incisor. Thirty-four myelinated nerve fibres are present: C-central core of myehnated nerve fibres; V~~lumen of blood vessel: OPodontoblast: Ct connective tissue cells. x 1400 Fig. 2. Electron

micrograph of the pulp of fully developed cat permanent incisor. One hundred five myelinated nerve fibres are present. M-myelinated nerve fibres. x 2000

and forty

Quantitation

of primary and permanent

teeth innervation

A.O.B. f.p. 678