Neuropeptide Y-immunoreactive primary afferents in the dental pulp and periodontal ligament following nerve injury to the inferior alveolar nerve in the rat

BRAIN RESEARCH ELSEVIER

Brain Research 712 (1996) 11-18

Research report

Neuropeptide Y-immunoreactive primary afferents in the dental pulp and periodontal ligament following nerve injury to the inferior alveolar nerve in the rat S. Wakisaka a,*, S.H. Youn a, j. Kato ", M. Takemura b, K. Kurisu

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Department of Oral Anatomy and Developmental Biology, Osaka UniL,ersity Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565, Japan b Second Department of Oral Anatomy, Osaka Uni~,ersity Facul~, of Dentist~', 1-8 Yamadaoka, Suita. Osaka 565, Japan Accepted 31 October 1995

Abstract

The distribution of neuropeptide Y (NPY)-immunoreactive (IR) primary afferents in the dental pulp and periodontal ligament of the rat mandible were examined following combined chronic constriction injury (CCI) of the inferior alveolar nerve (IAN) and sympathectomy of the superior cervical ganglion (SCG). NPY-IR nerve fibers were observed around the blood vessels in the trigeminal ganglion, dental pulp and periodontal ligament in normal animals. Following combined CCI of the IAN and sympathectomy of SCG (SCGx), perivascular NPY-IR nerve fibers originating from SCG disappeared completely, but many NPY-IR nerve fibers coming from the trigeminal ganglion appeared in the dental pulp and periodontal ligament. In the molar dental pulp, thick NPY-IR nerve fibers were observed within the nerve bundle, and some thin NPY-IR nerve fibers ran towards the odontoblast layer; very few NPY-IR nerve fibers were observed in the incisor pulp. In the periodontal ligament of molar, thick NPY-IR nerve fibers appeared at the alveolar part following combined CCI of IAN and SCGx. In the lingual portion of the periodontal ligament of the incisor, many thick NPY-IR nerve fibers were observed. These occasionally showed a tree-like appearance, resembling immature Ruffini endings; slowly adapting mechanoreceptors. The present results indicate that periodontal mechanoreceptors are among the main targets of injury-evoked NPY following IAN injury. Keywords: Nerve injury; Inferior alveolar nerve; Neuropeptide Y; Ruffini ending; Periodontal ligament; Mechanoreceptor; Rat

1. Introduction

Peripheral nerve injuries evoke alterations in the levels of several neurochemical markers. For example, substance P (SP) and calcitonin gene-related peptide (CGRP) are down-regulated in injured neurons [2,3,19,23,29]. In contrast, vasoactive intestinal polypeptide (VIP) and galanin (GAL) are up-regulated following peripheral nerve injury [2,16,22,29,38]. In addition to VIP and GAL, we previously reported marked increases in the levels of neuropeptide Y (NPY), a 36-amino acid polypeptide member of the pancreatic peptide family [34] which is distributed unevenly in both central and peripheral nervous systems [1,8,9,11,14,20], in the laminae I I I - V of the spinal dorsal horn following injury of the rat sciatic nerve [42,43]. Cell

* Corresponding author. wakisaka(cr dent .osaka-u. ac.j p

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size analysis revealed injury-evoked NPY-like immunoreactivity (LI) in the medium- to large-sized dorsal root ganglion (DRG) cells [13,42,43], suggesting that A/3 and A 6 neurons, or mechanoreceptors a n d / o r proprioceptors may be involved in the observed alterations in NPY level. We also reported that the sympathetic nervous system has no or very little role in the induction of NPY in the primary sensory neurons following nerve injury [45]. Fris~n et al. [13] reported that NPY-IR nerve fibers accumulated within the neuroma following tight ligation and transection of the rat sciatic nerve. Their findings indicated that nerve injury evokes de novo synthesis of NPY in the DRG and that the injury-evoked NPY is transported in an anterograde manner. The peripheral targets of injury-evoked NPY, however, remain obscure. Combined tight ligation and transection is the most widely used method to study the alterations in levels of neurochemicals following peripheral nerve injury. Such nerve injury completely interrupts axonal transport and

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does not permit nerve regeneration. Although the effect of axotomy is severe and changes in the levels of neurochemical synthesis can be detected easily, it is impossible to examine the alterations in levels of neurochemicals at sites distal to the site of nerve injury. In contrast, crush injury and loose ligation block the axonat transport only partially and allow nerve regeneration. Therefore, crush injury and loose ligation are suitable methods of nerve injury to study nerve regeneration. The same alteration in level of NPY-IR occurs in the trigeminal ganglion and trigeminal sensory nuclear complex following peripheral axotomy of the peripheral branches of the mandibular nerve or dental injury [17,26,44]. The inferior alveolar nerve (IAN) mainly innervates the dental pulp, periodontal ligament, oral mucosa and periosteum of the lower jaw, where the sympathetic nerve fibers come from the superior cervical ganglion (SCG). If A/3 and A 6 neurons in the trigeminal ganglion begin the synthesis of NPY in response to IAN injury, peripheral mechanoreceptors, such as Ruffini endings in the periodontal ligament, might display NPY-LI following IAN injury. Most NPY-IR nerve fibers belong to the sympathetic nerves in the peripheral nervous system under normal conditions [11,20], and sympathectomy of SCG (SCGx) abolishes NPY-IR nerve fibers in the IAN territory including the dental pulp [36]. In the present study, we investigated the distribution of NPY-IR primary afferents in the dental pulp and periodontal ligament of rat mandible following chronic constriction injury (CCI) of the IAN combined with SCGx to eliminate sympathetic NPY-IR nerve fibers.

2. Materials and methods

2.1. Animals and surgery A total of 8 Sprague-Dawley rats, weighing 200-250 g at the time of initial surgery, were divided into two groups and treated as follows; group I (n = 2) animals were sacrificed without undergoing any surgical procedures as normal controls, while those in group II (n = 6) underwent combined unilateral CC! of the IAN and SCGx. Surgical sympathectomy of SCG was performed under chloral hydrate anesthesia (400 m g / k g , i.p., supplemented as necessary) 5 days prior to IAN injury. The success of SCGx was confirmed by a decrease in the size of the ipsilateral pupil. For CCI of the IAN, animals received chloral hydrate anesthesia, an incision was made on the buccal skin, and the masseter muscle was torn and retracted to expose the buccal surface of the mandibular ramus ipsilateral to SCGx. A small amount of mandibular bone was removed with the aid of a dental drill to expose a segment of approximately 5 mm of the IAN within the mandibular canal. One loose ligation was placed around the IAN using 5-0 chromic gut. Sutures were made in anatomical layers.

2.2. Tissue preparation and immunohistochemistry Fourteen days after CCI of the IAN, when the effect of IAN injury reached its maximal level in the trigeminal ganglion [46], rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 0.02 M phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The mandibles and trigeminal ganglia were removed, postfixed in the same fixative overnight at 4°C, and soaked in PBS containing 20% sucrose overnight at 4°C. The mandibles were then decalcified with 7.5% ethylene diamine tetraacetic acid (EDTA) in 0.1 M phosphate buffer (pH 7.4) for 4 - 6 weeks at 4°C with gentle shaking. Decalcifying solution was changed at least 3 times a week. Following cryoprotection in 20% sucrose/PBS overnight, mandibles were sectioned longitudinally or transversely on a cryostat at a thickness of 30 p~m. The trigeminal ganglion was cut into 20 /xm sections horizontally. Sections were thawmounted onto poly-L-lysine-subbed glass slides, air-dried and processed for immunohistochemistry. An indirect immunofluorescence method was used for staining of the trigeminal ganglion. Sections were rinsed several times in PBS and incubated with rabbit anti-NPY antiserum (1:1000; Peninsula Lab., Belmont, CA) for 18 h at room temperature. Following several rinses in PBS, they were incubated with fluorescein isothiocyanate (FITC)conjugated anti-rabbit IgG (1:500; Cappei, West Chester, PA) for 90 min at 37°C in a moist chamber. Sections were rinsed, coverslipped with a mixture of glycerol-PBS (2:1) and viewed under a Nikon fluorescence microscope. The labeled streptavidin-biotin method was applied to the mandible sections. Following incubation with methanol containing 0.3% H202 for 10 min to block endogenous peroxidase activity, sections were treated with PBS containing 3% normal swine serum (NSS; Dako, Denmark) and 1% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) to block non-specific staining. They were then incubated with rabbit anti-NPY antiserum (1:5000) for 18 h at room temperature. Some sections were incubated with rabbit anti-protein gene product 9.5 (PGP 9.5; 1:700(I; Uitraclone, Cambridge, UK) to identify all neuronal structures [15,18,35]. Sections were incubated with biotinylated swine anti-rabbit IgG (1:300; Dakopatts, Denmark) for 90 min and then with horseradish peroxidase (HRP)-conjugated streptavidin (1:600; Dakopatts). All dilutions were made with PBS containing 1% NSS, 1% BSA and 0.1% Triton X-100. HRP was developed with a mixture of 0.04% diaminobenzidine tetrahydrochloride (DAB) and 0.01% H202 in 0.05 M Tris-HCl buffer (pH 7.6) with nickel ammonium sulfate intensification. Sections were counterstained with methyl green, dehydrated and coverslipped with Permount (Fisher Scientific, N J, USA). To examine the correlation between NPY and PGP 9.5, a double immunofluorescence method was applied. Sections were rinsed several times in PBS and incubated with

S. Wakisaka et al. / Brain Research 712 (1996) 11-18

a mixture of m o u s e m o n o c l o n a l a n t i - P G P 9.5 (1:5000; Ultraclone) and rabbit p o l y c l o n a l a n t i - N P Y (1:1000) antibodies for 18 h at r o o m temperature. Sections w e r e rinsed in P B S several times and incubated with biotinylated anti-mouse lgG (1:100; Vector) and subsequently with F I T C - c o n j u g a t e d streptavidin (1:50; Dakopatts) for 90 min each at r o o m temperature. N P Y was visualized by incubation with lissamine r h o d a m i n e ( L R S C ) - c o n j u g a t e d antirabbit IgG (1:500; Jackson I m m u n o R e s e a r c h ) for 90 min at 37°C in a h u m i d atmosphere. Sections w e r e coverslipped with a mixture o f P B S - g l y c e r o l , o b s e r v e d under a Nikon f l u o r e s c e n c e m i c r o s c o p e using B2 excitation filter for F I T C and G excitation filter for L R S C and photographed using K o d a k Tri X-pan film ( A S A 400).

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Specificity of primary antiserum against N P Y was tested by preabsorption using normal S C G . Preabsorption of the primary antiserum with an excess (10 /xM of diluted antiserum) of porcine N P Y (Sigma) c o m p l e t e l y eliminated the immunostaining. Specificity of anti-PGP 9.5 antiserum was verified by preabsorption in a previous study using the s a m e primary antiserum [15].

3. Results 3.1. T r i g e m i n a l g a n g l i o n

In the normal trigeminal ganglion (group I), s o m e N P Y IR nerve fibers were observed around the blood vessels,

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P

b

d

Fig. 1. Photomicrographs showing NPY-LI (a-d, f) and PGP 9.5-LI (e) in the dental pulp of the first molar (a) and incisor (c) of a normal rat and first molar (b, e, f) and incisor (d) from a rat following CCI of the IAN and SCGx. In the normal molar pulp (a), thin varicose NPY-IR nerve fibcrs formed a network around the blood vessel. Following CCI of the IAN and SCGx (b), NPY-IR nerve fibers arc observed within the nervc bundle (arrows); some thin NPY-IR nerve fibers (arrowhead) run towards the odontoblast layer (OD). In the normal incisor pulp, many NPY-IR nerve fibers run along the bh)od vessels (c), but following CCI of the IAN and SCGx, very few NPY-IR nerve fibers run along the blood vessels (arrowhead). Double immunostaining shows some PGP 9.5-IR nerve fibers (arrows in e) also exhibiting NPY-LI following CCI and SCGx (arrows in f). Scale bars: l(tl) ~m for a-f.

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but no NPY-IR ganglionic cells were detected. Following combined CCI of the IAN and SCGx, no NPY-IR nerve fibers were detected and many medium- to large-sized ganglion cells located in the mandibular division showed NPY-LI (data not shown).

3.2. Dental pulp In animals of group I, most NPY-IR nerve fibers were observed around the blood vessels of the molar and incisor pulp. In the molar pulp, fine NPY-IR nerve fibers formed a varicose network arrangement around the blood vessels (Fig. la). Following combined CCI of the IAN and SCGx, relatively thick NPY-IR nerve fibers with irregular swellings were observed within the nerve bundle; some thin NPY-IR nerve fibers ran towards the odontoblast (Fig. l b ) and they never formed a network arrangement. In normal incisor pulp, NPY-IR nerve fibers ran along the blood vessels (Fig. lc), and occasionally, very thin NPY-IR nerve fibers formed a network. Following CCI and SCGx, very few NPY-IR nerve fibers were observed along the blood vessels (Fig. ld). Double immunofluorescence analysis revealed that not all PGP 9.5-IR nerve fibers showed NPY-LI (Fig. le, f).

3.3. Periodontal ligament In the periodontal ligament of normal rats, fine varicose NPY-IR nerve fibers were associated with the blood ves-

sels (Fig. 2a) and these often formed a network. Following combined CCI and SCGx, numerous thick smooth-surfaced NPY-IR nerve fibers were observed in bundles at the alveolar side of the periodontal ligament and these were separated from the blood vessels. Thick NPY-IR nerve fibers entering the dental pulp along the blood vessels were also detected (Fig. 2b). In the periodontal ligament of the incisor of normal animals, the distribution and terminal morphology of PGP 9.5-IR nerve fibers were quite different between the lingolateral and labial portions. At the lingual portion, PGP 9.5-IR nerve bundles entered the lingual periodontal ligament; they ramified in a dendritic fashion and formed expanded terminals. The terminal morphology of these fibers was similar to that of Ruffini endings. They were restricted to the middle areas of the alveolar-related part of the periodontal ligament (Fig. 3a). Nerve fibers showing NPY-LI were rarely observed at the alveolar-related part of the lingual incisor periodontal ligament (Fig. 3b). Following CCI and SCGx, PGP 9.5-IR nerve fibers showed an irregular appearance, and no apparent Ruffini endings were observed (Fig. 3c); occasionally, thick PGP 9.5-IR nerve fibers showed a tree-like appearance. Many thick smooth-surfaced NPY-IR nerve fibers were observed in the nerve bundles (Fig. 3d). Some of these appeared near the tooth-related part of the lingual periodontal ligament (Fig. 3d). They ramified in a dendritic fashion, but less expanded than normal mature Ruffini endings (Fig. 3e, f); their terminals were expanded approximately 2 0 - 4 0 /xm.

Fig. 2. Photomicrographs of NPY-LI in the molar periodontal ligament from a normal rat (a) and from a rat with CCI and SCGx (b). Fine beaded NPY-IR nerve fibers are restricted around the blood vessels, and penetrate into the dental pulp (a). Following CCI of the IAN and SCGx (b), numerous NPY-IR nerve fibers are observed in bundles (arrowheads) at the alveolar portion of the periodontal ligament; some of these have no apparent relation with thc blood vessels. D, dentin. Scale bars: 100 ~m for a and b.

S. Wakisaka et al. /Brain Research 712 (1996) 11-18

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Fig. 3. Photomicrographs showing PGP 9.5-LI (a, c, g) and NPY-LI (b, d-f, h) at the lingual portion of the longitudinally sectioned incisor periodontal ligament of normal rats (a, b) and those following CCI of the IAN and removal of SCG (c-h). PGP 9.5-IR nerve fibers form Ruffini endings in the center of the alveolar-related part of the lingual portion of the periodontal ligament in normal animals (a); NPY-IR nerve fibers are rarely observed in the alveolar-related part (b). Following CCI of the IAN with SCGx, thick PGP 9.5-IR nerve fibers show an irregular appearance (c). Many thick smooth-surfaced NPY-IR nerve fibers are observed within the nerve bundle (arrow); some of them are localized near the tooth-related part (d). e shows a higher magnification of the boxed area in d. Two dendritic NPY-IR terminals show a tree-like appearance (arrowheads in e). More complex NPY-IR nerve terminals display a knob-like appearance (f). Double immunostaining shows that most PGP 9.5-IR nerve fibers (g) also display NPY-LI (h). AR, alveolar-related part; TR, tooth-related part; D, dentin. Scale bars: 50 /~m for a-f; 100 /.tm for g and h.

D o u b l e i m m u n o s t a i n i n g r e v e a l e d that m o s t P G P 9 . 5 - I R n e r v e f i b e r s s h o w e d N P Y - L I (Fig. 3g, h). A t the labial p o r t i o n o f the i n c i s o r p e r i o d o n t a l l i g a m e n t , P G P 9 . 5 - I R n e r v e f i b e r s ran a l o n g the a l v e o l a r side a n d t e r m i n a t e d as free e n d i n g s ( d a t a not s h o w n ) . T h i n b e a d e d N P Y - I R n e r v e fibers w e r e o b s e r v e d a r o u n d the b l o o d v e s s e l s (Fig. 4a). F o l l o w i n g loose l i g a t i o n of I A N a n d

r e m o v a l o f S C G , no N P Y - I R n e r v e f i b e r s w e r e o b s e r v e d (Fig. 4b).

4. Discussion In the p r e s e n t i m m u n o h i s t o c h e m i c a l study, we first d e m o n s t r a t e d the d i s t r i b u t i o n o f i n j u r y - e v o k e d N P Y in the

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Fig. 4. Photomicrographs of NPY-L1 in the labial portion of the incisor periodontal ligament. Fine varicose NPY-IR nerve fibers are observed around the blood vessels (arrowhead in a). No nerve fibers display NPY-IR following CCI of the IAN and SCGx (b). A B E ameloblast layer; PDL, pcriodontal ligament. Scale bars: 100 ,ttm for a and b.

dental pulp and periodontal ligament, one of the receptive fields of the IAN, following CCI of the IAN combined with SCGx. Many studies of the alterations in levels of neurochemical markers following nerve injuries have been carried out using experimental nerve injury to the sciatic nerve since surgery on this nerve is relatively easy. To examine the changes in levels of NPY in the periphery following nerve injury, sympathectomy was necessary to destroy all sympathetic NPY-IR nerve fibers. It is very difficult to destroy the sympathetic neurons in the receptive fields of the sciatic nerve completely. In contrast, SCGx was relatively easy. Therefore, we chose CCI of the IAN as a model of nerve injury to detect the distribution of NPY-IR nerve fibers distal to the site of nerve injury, although decalcification was necessary to detect NPY-IR nerve fibers in the dental pulp and periodontal tissues. We believe that all NPY-IR nerve fibers observed in the animals with combined CCI to IAN and SCGx (group II) were induced by nerve injury to the IAN, and were primary afferents. We did not carry out temporal analysis on regeneration of the injured nerve fibers in the pulp or periodontal ligament in the present study. Our previous study [40] revealed that, following resection of the IAN, SP-IR nerve fibers in the dental pulp disappeared completely and regenerating SP-IR nerve fibers were observed 14 days post-injury. Following application of HRP to the rat molar dental pulp after IAN resection, HRP-labeled trigeminal ganglion cells were observed 3 days after injury [33]. PGP 9.5-IR nerve fibers in the periodontal ligament following IAN injury showed an irregular appearance. It is known that regenerating nerve fibers show irregular swellings and occasionally the number of these nerve fibers increases transiently. These findings suggested that the fibers observed 14 days after IAN injury in the present study may have been regenerating nerve fibers. The sensory modality of primary afferent neurons innervating the dental pulp has been considered to be pain. Immunohistochemical studies revealed that dental pulp

receives rich nerve supply showing SP- and CGRP-LI [25,30,37,39,41]. However, cell-size analysis of trigeminal ganglion cells retrogradely labeled from tooth pulp revealed that labeled cells were relatively large [31]. Histochemical analysis indicated that trigeminal ganglion cells labeled with retrograde tracer applied to the tooth pulp contained chemical markers such as carbonic anhydrase and GM1 ganglioside [12,32]. Moreover, electrophysiological studies showed that some intradental nerve units have conduction velocities within the A/3 range [6,10]. These lines of evidence indicated that some tooth pulp primary neurons have large cell bodies, probably with A/3 neurons. Our previous studies revealed that IAN transection or dental injury induced the appearance of NPY-IR in medium- to large-sized cells in the trigeminal ganglion [17,26,46]. If the injury-evoked NPY was transported anterogradely from the trigeminal ganglion to the nerve terminals of A/3 and A 6 neurons, many nerve fibers in the dental pulp of the animals in group II would have shown NPY-EI. As shown by double immunostaining, a small number of PGP 9.5-IR nerve fibers in the dental pulp displayed NPY-LI, suggesting that NPY-IR primary afferents are a small subpopulation of pulpal nerve fibers following IAN injury. In contrast to the dental pulp, many NPY-IR primary afferents were observed in the alveolar-related part of the lingual portion of the incisor periodontal ligament. Recently, Ruffini endings, slowly-adapting mechanoreceptors [4,7], have been found in the periodontal ligament of rodent molars and incisors [5,21,24,27,28]. Ruffini endings are easily distinguished by their morphological characteristics; the endings are repeatedly branched in a dendritic fashion with each terminal twig being expanded. In the periodontal ligament of the rodent incisor, they are restricted to the middle areas of the alveolar-related part of the lingual region, and can be immunostained for some neuron-specific proteins, such as neurofilament protein, glia-specific S-100 protein and PGP 9.5 [21,24,27,28]. However, to our knowledge, no neuropeptides have been

S. Wakisaka et aL / Brain Research 712 (19961 11-18

demonstrated in the Ruffini endings in normal animals. Following combined CCI of the IAN and SCGx, NPY-IR nerve fibers appeared, and these ramified in a dendritic fashion. Their terminal morphology was different from that of mature Ruffini endings immunostained by antiserum against PGP 9.5 in normal animals, but was similar to that of immature Ruffini endings observed in 4- to 10-day-old rats [24]. Nerve injury is known to induce degeneration distal to the site of injury, followed by regeneration. Therefore, the terminal morphology of NPY-IR nerve fibers in the alveolar-related part of the rat incisor periodontal ligament may represent regenerating features of Ruffini endings, although terminals of NPY-IR nerve fibers were located near the tooth-related part, while normal Ruffini endings were localized in the middle areas of the alveolarrelated part. Further electron microscopic analysis is required to determine whether terminals of NPY-IR nerve fibers show the typical terminal morphology of Ruffini endings. Double immunostaining revealed that most PGP 9.5-IR nerve fibers exhibited NPY-LI, suggesting that the periodontal nerve fibers, presumably Ruffini endings, are the main target of injury-evoked NPY following IAN injury. The functional significance of injury-evoked NPY has yet to be determined. Previously, we speculated from cell-size analysis of injury-evoked NPY that NPY in primary afferents may be involved in the regeneration of mechanoreceptors and/or proprioceptors, or in sensory transmission of A/3 or A 6 neurons following nerve injury [26,42-44,46]. The present study demonstrated that mechanoreceptors are among the targets of injury-evoked NPY and that injury-evoked NPY-IR may participate in the regeneration of myelinated nerve fibers since NPY-IR was present in what were presumed to be regenerating Ruffini endings. Further analysis of the relationship between the regenerative process of mechanoreceptors and expression of NPY is necessary.

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Acknowledgements This study was partly supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Science and Culture of Japan (07671968).

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