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NADPH-diaphorase activity in nerves and Schwann cells in the periodontal ligament of rat incisor teeth
Archives of Oral Biology 43 (1998) 167±171
ARCHIVES OF ORAL BIOLOGY
Short communication
NADPH-diaphorase activity in nerves and Schwann cells in the periodontal ligament of rat incisor teeth Hiroyuki Ichikawa *, Tomosada Sugimoto Second Department of Oral Anatomy, Okayama University Dental School, Okayama, Japan
Abstract The lingual portion of the incisor periodontal ligament demonstrated activity for nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase. Schwann cells surrounding Runi-like endings coexpressed NADPH-diaphorase activity and immunoreactivity for inducible nitric oxide synthase. NADPH-diaphorase-positive nerve ®bres which coexpressed immunoreactivity for neuronal nitric oxide synthase were in contact with Schwann cells surrounding Runi-like endings or terminated as free nerve endings. Neural NADPH-diaphorase activity could not be found in the tissues covering the labial portion of incisor tooth root. It is possible that nitric oxide in Schwann cells and nerves has functions speci®c to the incisor periodontal ligament. # 1998 Elsevier Science Ltd. All rights reserved Key words: Nicotinamide adenosine dinucleotide phosphate-diaphorase, Periodontal ligament, Nitric oxide synthase, Runi-like ending, Free nerve ending, Incisor, Rat
NADPH-diaphorase, a histochemical marker for nitric oxide synthase, occurs in the central and peripheral nervous systems (Aimi et al., 1991; Alm et al., 1995; Hisa et al., 1996; Kerezoudis et al., 1993b; Morris et al., 1993; Paakkari and Lindsberg, 1995; Vincent and Kimura, 1992). In the trigeminal ganglion, it is localized in small to medium-sized primary sensory neurones (Aimi et al., 1991; Alm et al., 1995; Kerezoudis et al., 1993b; Morris et al., 1993). These neurones also coexpress immunoreactivities for substance P and CGRP (Aimi et al., 1991). Because these two are considered to be putative transmitters for nociception in primary sensory neurones, NADPH-diaphorase-positive trigeminal neurones may include primary nociceptors. Recently, it has been demonstrated that NADPH-diaphorase in neurones is neuronal nitric oxide synthase, one of the enzyme's isoforms (Hisa et al., 1996; Paakkari and Lindsberg, 1995). In oral tissues, inhibition of this neuronal isoform reportedly had eects on basal blood ¯ow and antidromic vasodi-
* To whom all correspondence should be addressed. Abbreviations: CGRP, calcitonin gene-related peptide, NADPH, nicotinamide adenosine dinucleotide phosphate. 0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved PII: S 0 0 0 3 - 9 9 6 9 ( 9 7 ) 0 0 0 9 9 - X
lation but not neurogenic plasma extravasation (Kerezoudis et al., 1993a; 1994). On the other hand, glial cells in the brainstem contain macrophage-type inducible nitric oxide synthase and NADPH-diaphorase (Galea et al., 1992; Paakkari and Lindsberg, 1995). Nitric oxide synthesized by the inducible synthase has been suggested to have various functions associated with host defence and plasticity in glial cells, and immunoregulatory roles of Schwann cells (Gold et al., 1996; Paakkari and Lindsberg, 1995). The periodontal ligament receives innervation from primary sensory neurones. Their receptors include free and Runi-like endings (Anderson et al., 1970; Byers, 1985; Byers and Dong, 1989; Byers et al., 1986; Sato et al., 1988; Silverman and Kruger, 1987; Wakisaka et al., 1985). Periodontal free nerve endings display substance P and CGRP immunoreactivities, and are thought to be nociceptors probably derived from small neurones in the trigeminal ganglion (Anderson et al., 1970; Byers, 1985; Byers and Dong, 1989; Silverman and Kruger, 1987; Wakisaka et al., 1985). Neurones with periodontal Runi-like endings have their cell bodies in the trigeminal mesencephalic nucleus or trigeminal ganglion (Byers, 1985; Byers and Dong, 1989). The receptors on the mesencephalic nucleus are
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H. Ichikawa, T. Sugimoto / Archives of Oral Biology 43 (1998) 167±171
thought to be involved in monitoring tooth movement and in the re¯ex control of mandibular movements during mastication (Byers et al., 1986). Runi-like endings of trigeminal neurones are mechanoreceptors that are thought to be activated by touch, pressure and movement of teeth during chewing, swallowing and speech. Axon terminals in Runi-like endings are covered with lamellar Schwann cells that are immunoreactive for glia-speci®c S100 protein (Sato et al., 1988). A previous study demonstrated that in the periodontal ligament of rat molar teeth these receptors were devoid of NADPH-diaphorase activity (Kerezoudis et al., 1993b). The structures and functions of periodontal ligaments are dierent in rat molar and incisor teeth, and NADPH-diaphorase activity has never been reported in the incisor periodontal ligament. We have now examined NADPHdiaphorase activity in the periodontal ligaments of rat incisor teeth in order to determine whether periodontal receptors utilize nitric oxide. The coexpression of NADPH-diaphorase activity with immunoreactivities for neuronal and inducible isoforms of nitric oxide synthase and S100, and the ultrastructure of inducible nitric oxide synthase-immunoreactive components were also investigated to characterize NADPH-diaphorasepositive pro®les. Eight adult male Sprague±Dawley rats (200±300 g) were used. Animals were anaesthetized with ether to the level at which respiration was markedly suppressed, and transvascularly perfused with 50 ml of isotonic saline (154 mM NaCl) followed by 500 ml of 4% formaldehyde in 0.1 M phosphate buer (pH 7.4). For three animals to be examined by electron microscopy, 0.05% glutaraldehyde was added to the ®xative. Mandibles containing incisor teeth were dissected and mineralized with 4.13% EDTA disodium salt in 0.1 M phosphate buer (pH 7.4) for 1 week at 48C. The tissues were soaked overnight in a phosphate-buered saline containing 20% sucrose, frozen-sectioned at 12 mm, and mounted on gelatin-coated glass slides. For NADPH-diaphorase histochemistry, sagittal sections were incubated with 0.1 M phosphate buer (pH 7.4) containing 0.1 mg/ml nitroblue tetrazolium (Sigma, U.S.A.) and 1.0 mg/ml b-NADPH (Sigma) for 2 hr at 378C. For the coexpression study, a double-immuno¯uorescence method was used. Sections were incubated with a mixture of rabbit anti-neuronal nitric-oxide synthase serum (1:1000; Chemicon International, Inc., U.S.A.) and mouse monoclonal anti-S100 antibody (1:1000; Sigma) or with a mixture of rabbit anti-inducible nitric-oxide synthase serum (1:1000; Santa Cruz Biotechnology Inc., U.S.A.) and mouse monoclonal anti-S100 antibody, followed by incubation with a mixture of lissamine rhodamine B chloride-conjugated donkey antirabbit IgG (1:400; Jackson
ImmunoResearch Labs, U.S.A.) and ¯uorescein isothiocyanate-conjugated donkey antimouse IgG (1:100; Jackson ImmunoResearch). Subsequent to photomicroscopy of immuno¯uorescent pro®les, the coverslips were removed, and the sections were stained for NADPH-diaphorase acitivity. For electron microscopy, unfrozen 50 mm-thick sagittal sections were cut with a Microslicer (Dosaka EM, Japan) and stained for inducible nitric-oxide synthase immunoreactivity with an avidin±biotin±horseradish peroxidase complex method. The sections were incubated with the primary antibody (1:20000) for 5 days at 48C followed by biotinylated goat antirabbit IgG and the avidin complex (Vector Laboratories, U.S.A.). Following diaminobenzidine reaction, these sections were post®xed in 1% osmium tetroxide in 0.1 M phosphate buer (pH 7.4), dehydrated through a graded series of alcohols, and embedded in Polybed 812. Ultrathin sections were examined after staining with lead citrate for 1 min. In control experiments, the primary antibodies were preabsorbed with appropriate proteins (50 mg/ml; Santa Cruz Biotechnology for neuronal and inducible synthases, Sigma for S100). No staining was observed in the controls. To examine whether the NADPH-diaphorase-positive ®bres observed were neural elements, the right inferior alveolar nerve was transected in one rat, and the mandible was stained for NADPH-diaphorase. At 7 days after the transection, virtually all positively stained ®bres had disappeared from the periodontal ligament of the mandibular teeth on the ipsilateral side. Thus, we consider that the NADPHdiaphorase-positive ®bres were nerve ®bres, and the term ``nerve ®bres'' is used throughout this paper. NADPH-diaphorase activity was observed in cells and nerve ®bres in the lingual portion of incisor periodontal ligament (Fig. 1A±C). These cells and ®bres were adjacent to the alveolar bone. The cells had various shapes, including round, oval and triangular, with or without processes (diameters of cells, 3±10 mm), and were aggregated in close apposition to nerve bundles and blood vessels (mean number 2S.E.M of positive cells/section = 126 217, n = 4). The reaction products for this enzyme were restricted to the cytoplasm. NADPH-diaphorase-positive ®bres were abundant in nerve bundles and had a ®ne, varicose appearance. Some positive nerve ®bres were also observed accompanying blood vessels. These ®bres left nerve bundles and blood vessels, and surrounded NADPH-diaphorase-positive cells (Fig. 1B) or terminated as free nerve endings (Fig. 1C). Nearly 30% (137/505) of NADPHdiaphorase-positive cells were seen in close contact with the positive nerve ®bres. The double-immuno¯uorescence method in combination with NADPH-diaphorase histochemistry revealed coexpression of NADPH-diaphorase activity
H. Ichikawa, T. Sugimoto / Archives of Oral Biology 43 (1998) 167±171
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Fig. 1. Photomicrographs of NADPH-diaphorase activity (A±D, G), inducible nitric oxide synthase (iNOS) immunoreactivity (E, I, J), S100-immunoreactivity (F) and neuronal nitric oxide synthase (nNOS) immunoreactivity (H) in the lingual portion of the incisor periodontal ligament. NADPH-diaphorase-positive cells have various shapes with or without processes (arrows in A, B). NADPHdiaphorase-positive nerve ®bres surround the positive cells (arrowheads in B) in close apposition to a blood vessel (bv in B) or terminate as free nerve endings (arrowheads in C). (D±F) and (G, H) are the same ®elds of views. NADPH-diaphorase-positive cells (arrows in D) coexpress both iNOS (arrows in E) and S100 immunoreactivities (arrows in F). The distribution of NADPH-diaphorase-positive nerve ®bres (arrowheads in G) is very similar to that of nNOS-immunoreactive nerve ®bres (arrowheads in H). Arrows in (G) and (H) indicate NADPH-diaphorase-positive cells (G) which are devoid of nNOS immunoreactivity (H). (I) and (J) show electron micrographs of a Runi-like ending obtained from adjacent sections. A terminal Schwann cell surrounding a Runi-like ending (s in I) contains dense reaction products for iNOS immunoreactivity (large arrowheads in I) and is covered with multiple layers of basal lamina (small arrows in I). Large arrows in (I) and (J) indicate the same short projection of the axoplasm. Clusters of small vesicles are seen at the base of the projection (small arrowheads in I and J). Scale bars: 50 mm (A±C), 100 mm (D±H) and 1 mm (I, J).
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with immunoreactivities for inducible and neuronal nitric oxide synthase and S100 in the periodontal ligament. All NADPH-diaphorase-positive cells coexpressed the inducible synthase and S100 immunoreactivities. In addition, all cells immunoreactive for inducible nitric oxide synthase coexpressed NADPH-diaphorase activity and S100 immunoreactivity (Fig. 1D±F). In nerve bundles, however, S100-immunoreactive Schwann cells were devoid of NADPHdiaphorase activity and inducible synthase immunoreactivity. NADPH-diaphorase-positive cells were always devoid of immunoreactivity for neuronal nitric oxide synthase. The distributions of NADPH-diaphorase-positive nerve ®bres and neuronal nitric oxide synthase-immunoreactive ones were identical (Fig. 1, G, H). No nerve ®bres immunoreactive for the inducible synthase were observed in the periodontal ligament. The coexpression study demonstrated that NADPHdiaphorase-positive cells and cells immunoreactive for inducible nitric oxide synthase had an identical distribution in the periodontal ligament of incisor teeth. Thus, inducible synthase-immunoreactive cells were examined by an immunoelectron-microscopic method to characterize NADPH-diaphorase-positive cells. The clusters of inducible synthase-immunoreactive cells in the periodontal ligament turned out to be Runi-like endings (Fig. 1, I). Terminal Schwann cells surrounding Runi-like endings contained numerous pinocytotic vesicles and some mitochondria, and formed lamellar sheets around the axoplasm. These Schwann cells were covered with multiple layers of basal lamina. The axoplasm was enriched with mitochondria (Fig. 1, I) and formed short projections that extended into the surrounding intercellular space (Fig. 1, J). Clusters of small vesicles were often seen at the base of the projections. It was the Schwann cell and its process that expressed immunoreactivity for inducible nitric oxide synthase in the Runi-like endings, while the axoplasm was devoid of it. At a higher magni®cation, the immunoreaction products were distributed over membranes of pinocytotic vesicles and the cytoplasm in Schwann cells. Neural NADPH-diaphorase activity could not be observed in the tissues covering the labial portion of the roots of incisor teeth. We demonstrate that the periodontal ligament of rat incisor teeth contains neural NADPH-diaphorase activity. NADPH-diaphorase-positive cells that coexpressed immunoreactivities for inducible nitric oxide synthase and S100 were distributed in the lingual portion of the ligament. Our electron-microscopic analysis for the inducible synthase immunoreactivity indicated that these NADPH-diaphorase-positive cells were identical to Schwann cells and their processes associated with Runi-like endings. This is supported by previous
®ndings that glial cells in the brainstem contained NADPH-diaphorase activity and immunoreactivity for inducible nitric oxide synthase (Galea et al., 1992; Paakkari and Lindsberg, 1995) and that Schwann cells in periodontal Runi-like endings exhibited S100 immunoreactivity (Sato et al., 1988). Because Schwann cells in nerve bundles were devoid of NADPH-diaphorase activity and the inducible synthase immunoreactivity, these cells are probably unable to synthesize nitric oxide. Thus, nitric oxide may be associated with the functions of Schwann cells speci®c to Runi-like endings surrounding the incisor periodontal ligament. We also demonstrate that nerve ®bres in the incisor periodontal ligament coexpress NADPH-diaphorase activity and immunoreactivity for neuronal nitric oxide synthase. Because both primary sensory neurones in the trigeminal ganglion and parasympathetic postganglionic neurones in the otic and pterygopalatine ganglia contain NADPH-diaphorase activity (Aimi et al., 1991; Alm et al., 1995; Morris et al., 1993; Kerezoudis et al., 1993b), the origin of these positively stained periodontal nerve ®bres is still unclear. However, their sensory nature is probably suggested by the distribution of their terminals; NADPH-diaphorase-positive nerve ®bres terminated as free nerve endings or were in contact with Schwann cells in Runi-like endings. This may be supported by our control ®ndings that virtually all NADPH-diaphorasepositive nerve ®bres in the incisor periodontal ligament disappeared after transection of the inferior alveolar nerve. The possibility that these nerve ®bres originate from the trigeminal mesencephalic nucleus is excluded, because primary sensory neurones in that nucleus are devoid of the enzyme activity (Vincent and Kimura, 1992) and because periodontal receptors from that nucleus other than Runi-like endings have not been reported (Byers et al., 1986). Thus, it can be deduced that NADPH-diaphorase-positive nerve ®bres in the periodontal ligament at least partly originate from the trigeminal ganglion. The coexpression of CGRP and substance P immunoreactivities by the NADPH-diaphorase-positive trigeminal cells (Aimi et al., 1991) and the demonstrated free nerve endings strongly suggest that at least some of the NADPH-diaphorase-positive trigeminal neurones are involved in nociception. It is possible that nitric oxide synthesized by trigeminal neurones is involved in sensory signal transduction (Paakkari and Lindsberg, 1995). On the other hand, the preferential distribution of NADPH-diaphorase activity and nitric oxide synthase immunoreactivity in the periodontal ligament of the incisor but not molar teeth may suggest that the neural nitric oxide is related to the speci®c tissue environment. Because the adult rodent incisor is continually erupting, the sensory endings in its periodontal ligament have to accommodate more
H. Ichikawa, T. Sugimoto / Archives of Oral Biology 43 (1998) 167±171
tissue reorganization than is the case for molars. Nitric oxide may thus be involved in the plasticity of sensory nerve endings in the rapidly erupting incisor periodontal ligament.
References Aimi, Y., Fujimura, M., Vincent, S. R., Kimura, H. (1991) Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat. J. Comp. Neurol. 30, 382±392. Alm, P., Uvelius, B., Ekstrom, J., Holmqvist, B., Larsson, B., Andersson, K-E. (1995) Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study. Histochem. J. 27, 819±831. Anderson, D. J., Hannam, A. G., Matthews, B. (1970) Sensory mechanisms in mammalian teeth and their supporting structures. Physiol. Rev 50, 171±195. Byers, M. R. (1985) Sensory innervation of periodontal ligament of rat molars consists of uncapsulated Runi-like mechanoreceptors and free nerve endings. J. Comp. Neurol. 231, 500±518. Byers, M. R., Dong, W. K. (1989) Comparison of trigeminal receptor location and structure in the periodontal ligament of dierent types of teeth from the rat, cat and monkey. J. Comp. Neurol. 279, 117±127. Byers, M. R., O'Connor, T. A., Martin, R. F., Dong, W. K. (1986) Mesencepharic trigeminal sensory neurons of cat; Axon pathways and structure of mechanoreceptive endings in periodontal ligament. J. Comp. Neurol. 250, 181±191. Galea, E., Feinstein, D. L., Reis, D. J. (1992) Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures. Proc. Natl. Acad. Sci. U.S.A. 89, 10945±10949. Gold, R., Zielasek, J., Kiefer, R., Toyka, K. V., Hartung, H. P. (1996) Secretion of nitrite by Schwann cells and its eect on T-cell activation. Cell. Immunol. 168, 69±77.
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Hisa, Y., Tadaki, N., Uno, T., Koike, S., Tanaka, M., Okamura, H., Ibata, Y. (1996) Nitrergic innervation of the rat larynx measured by nitric oxide synthase immunohistochemistry and NADPH-diaphorase histochemistry. Ann. Otol. Rhinol. Laryngol. 105, 550±554. Kerezoudis, N. P., Olgart, L., Edwall, L. (1993a) Dierential eects of nitric oxide synthasis inhibition on basal blood ¯ow and antidromic vasodilation in rat oral tissues. Eur. J. Pharmacol. 241, 209±219. Kerezoudis, N. P., Olgart, L., Edwall, L. (1994) Involvement of substance P but not nitric oxide or calcitonin gene-related peptide in neurogenic plasm extravasation in rat incisor pulp and lip. Arch. Oral. Biol. 39, 769±774. Kerezoudis, N. P., Olgart, L., Fried, K. (1993b) Localization of NADPH-diaphorase activity in the dental pulp, periodontium and alveolar bone of the rat. Histochemistry 100, 319±322. Morris, R., Southam, E., Gittins, S. R., Garthwaite, J. (1993) NADPH-diaphorase staining in autonomic and somatic cranial ganglia of the rat. NeuroReport 4, 65±68. Paakkari, I., Lindsberg, P. (1995) Nitric oxide in the central nervous system. Ann. Med. 27, 369±377. Sato, O., Maeda, T., Kobayashi, S., Iwanaga, T., Fujita, T., Takahashi, Y. (1988) Innervation of periodontal ligament and dental pulp in rat incisors. An immunohistochemical investigation of neuro®lament protein and glia speci®c S100 protein. Cell Tiss. Res. 251, 13±21. Silverman, J. D., Kruger, L. (1987) An interpretation of dental innervation based upon the pattern of calcitonin generelated peptide (CGRP) immunohistochemistry. Somatosen. Res. 5, 157±175. Vincent, S. R., Kimura, H. (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755± 784. Wakisaka, S., Nishikawa, S., Ichikawa, H., Matsuo, S., Takano, Y., Akai, M. (1985) The distribution and origin of substance P-like immunoreactivity in the rat molar pulp and periodontal tissues. Arch. oral. Biol. 30, 813±818.
ARCHIVES OF ORAL BIOLOGY
Short communication
NADPH-diaphorase activity in nerves and Schwann cells in the periodontal ligament of rat incisor teeth Hiroyuki Ichikawa *, Tomosada Sugimoto Second Department of Oral Anatomy, Okayama University Dental School, Okayama, Japan
Abstract The lingual portion of the incisor periodontal ligament demonstrated activity for nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase. Schwann cells surrounding Runi-like endings coexpressed NADPH-diaphorase activity and immunoreactivity for inducible nitric oxide synthase. NADPH-diaphorase-positive nerve ®bres which coexpressed immunoreactivity for neuronal nitric oxide synthase were in contact with Schwann cells surrounding Runi-like endings or terminated as free nerve endings. Neural NADPH-diaphorase activity could not be found in the tissues covering the labial portion of incisor tooth root. It is possible that nitric oxide in Schwann cells and nerves has functions speci®c to the incisor periodontal ligament. # 1998 Elsevier Science Ltd. All rights reserved Key words: Nicotinamide adenosine dinucleotide phosphate-diaphorase, Periodontal ligament, Nitric oxide synthase, Runi-like ending, Free nerve ending, Incisor, Rat
NADPH-diaphorase, a histochemical marker for nitric oxide synthase, occurs in the central and peripheral nervous systems (Aimi et al., 1991; Alm et al., 1995; Hisa et al., 1996; Kerezoudis et al., 1993b; Morris et al., 1993; Paakkari and Lindsberg, 1995; Vincent and Kimura, 1992). In the trigeminal ganglion, it is localized in small to medium-sized primary sensory neurones (Aimi et al., 1991; Alm et al., 1995; Kerezoudis et al., 1993b; Morris et al., 1993). These neurones also coexpress immunoreactivities for substance P and CGRP (Aimi et al., 1991). Because these two are considered to be putative transmitters for nociception in primary sensory neurones, NADPH-diaphorase-positive trigeminal neurones may include primary nociceptors. Recently, it has been demonstrated that NADPH-diaphorase in neurones is neuronal nitric oxide synthase, one of the enzyme's isoforms (Hisa et al., 1996; Paakkari and Lindsberg, 1995). In oral tissues, inhibition of this neuronal isoform reportedly had eects on basal blood ¯ow and antidromic vasodi-
* To whom all correspondence should be addressed. Abbreviations: CGRP, calcitonin gene-related peptide, NADPH, nicotinamide adenosine dinucleotide phosphate. 0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved PII: S 0 0 0 3 - 9 9 6 9 ( 9 7 ) 0 0 0 9 9 - X
lation but not neurogenic plasma extravasation (Kerezoudis et al., 1993a; 1994). On the other hand, glial cells in the brainstem contain macrophage-type inducible nitric oxide synthase and NADPH-diaphorase (Galea et al., 1992; Paakkari and Lindsberg, 1995). Nitric oxide synthesized by the inducible synthase has been suggested to have various functions associated with host defence and plasticity in glial cells, and immunoregulatory roles of Schwann cells (Gold et al., 1996; Paakkari and Lindsberg, 1995). The periodontal ligament receives innervation from primary sensory neurones. Their receptors include free and Runi-like endings (Anderson et al., 1970; Byers, 1985; Byers and Dong, 1989; Byers et al., 1986; Sato et al., 1988; Silverman and Kruger, 1987; Wakisaka et al., 1985). Periodontal free nerve endings display substance P and CGRP immunoreactivities, and are thought to be nociceptors probably derived from small neurones in the trigeminal ganglion (Anderson et al., 1970; Byers, 1985; Byers and Dong, 1989; Silverman and Kruger, 1987; Wakisaka et al., 1985). Neurones with periodontal Runi-like endings have their cell bodies in the trigeminal mesencephalic nucleus or trigeminal ganglion (Byers, 1985; Byers and Dong, 1989). The receptors on the mesencephalic nucleus are
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H. Ichikawa, T. Sugimoto / Archives of Oral Biology 43 (1998) 167±171
thought to be involved in monitoring tooth movement and in the re¯ex control of mandibular movements during mastication (Byers et al., 1986). Runi-like endings of trigeminal neurones are mechanoreceptors that are thought to be activated by touch, pressure and movement of teeth during chewing, swallowing and speech. Axon terminals in Runi-like endings are covered with lamellar Schwann cells that are immunoreactive for glia-speci®c S100 protein (Sato et al., 1988). A previous study demonstrated that in the periodontal ligament of rat molar teeth these receptors were devoid of NADPH-diaphorase activity (Kerezoudis et al., 1993b). The structures and functions of periodontal ligaments are dierent in rat molar and incisor teeth, and NADPH-diaphorase activity has never been reported in the incisor periodontal ligament. We have now examined NADPHdiaphorase activity in the periodontal ligaments of rat incisor teeth in order to determine whether periodontal receptors utilize nitric oxide. The coexpression of NADPH-diaphorase activity with immunoreactivities for neuronal and inducible isoforms of nitric oxide synthase and S100, and the ultrastructure of inducible nitric oxide synthase-immunoreactive components were also investigated to characterize NADPH-diaphorasepositive pro®les. Eight adult male Sprague±Dawley rats (200±300 g) were used. Animals were anaesthetized with ether to the level at which respiration was markedly suppressed, and transvascularly perfused with 50 ml of isotonic saline (154 mM NaCl) followed by 500 ml of 4% formaldehyde in 0.1 M phosphate buer (pH 7.4). For three animals to be examined by electron microscopy, 0.05% glutaraldehyde was added to the ®xative. Mandibles containing incisor teeth were dissected and mineralized with 4.13% EDTA disodium salt in 0.1 M phosphate buer (pH 7.4) for 1 week at 48C. The tissues were soaked overnight in a phosphate-buered saline containing 20% sucrose, frozen-sectioned at 12 mm, and mounted on gelatin-coated glass slides. For NADPH-diaphorase histochemistry, sagittal sections were incubated with 0.1 M phosphate buer (pH 7.4) containing 0.1 mg/ml nitroblue tetrazolium (Sigma, U.S.A.) and 1.0 mg/ml b-NADPH (Sigma) for 2 hr at 378C. For the coexpression study, a double-immuno¯uorescence method was used. Sections were incubated with a mixture of rabbit anti-neuronal nitric-oxide synthase serum (1:1000; Chemicon International, Inc., U.S.A.) and mouse monoclonal anti-S100 antibody (1:1000; Sigma) or with a mixture of rabbit anti-inducible nitric-oxide synthase serum (1:1000; Santa Cruz Biotechnology Inc., U.S.A.) and mouse monoclonal anti-S100 antibody, followed by incubation with a mixture of lissamine rhodamine B chloride-conjugated donkey antirabbit IgG (1:400; Jackson
ImmunoResearch Labs, U.S.A.) and ¯uorescein isothiocyanate-conjugated donkey antimouse IgG (1:100; Jackson ImmunoResearch). Subsequent to photomicroscopy of immuno¯uorescent pro®les, the coverslips were removed, and the sections were stained for NADPH-diaphorase acitivity. For electron microscopy, unfrozen 50 mm-thick sagittal sections were cut with a Microslicer (Dosaka EM, Japan) and stained for inducible nitric-oxide synthase immunoreactivity with an avidin±biotin±horseradish peroxidase complex method. The sections were incubated with the primary antibody (1:20000) for 5 days at 48C followed by biotinylated goat antirabbit IgG and the avidin complex (Vector Laboratories, U.S.A.). Following diaminobenzidine reaction, these sections were post®xed in 1% osmium tetroxide in 0.1 M phosphate buer (pH 7.4), dehydrated through a graded series of alcohols, and embedded in Polybed 812. Ultrathin sections were examined after staining with lead citrate for 1 min. In control experiments, the primary antibodies were preabsorbed with appropriate proteins (50 mg/ml; Santa Cruz Biotechnology for neuronal and inducible synthases, Sigma for S100). No staining was observed in the controls. To examine whether the NADPH-diaphorase-positive ®bres observed were neural elements, the right inferior alveolar nerve was transected in one rat, and the mandible was stained for NADPH-diaphorase. At 7 days after the transection, virtually all positively stained ®bres had disappeared from the periodontal ligament of the mandibular teeth on the ipsilateral side. Thus, we consider that the NADPHdiaphorase-positive ®bres were nerve ®bres, and the term ``nerve ®bres'' is used throughout this paper. NADPH-diaphorase activity was observed in cells and nerve ®bres in the lingual portion of incisor periodontal ligament (Fig. 1A±C). These cells and ®bres were adjacent to the alveolar bone. The cells had various shapes, including round, oval and triangular, with or without processes (diameters of cells, 3±10 mm), and were aggregated in close apposition to nerve bundles and blood vessels (mean number 2S.E.M of positive cells/section = 126 217, n = 4). The reaction products for this enzyme were restricted to the cytoplasm. NADPH-diaphorase-positive ®bres were abundant in nerve bundles and had a ®ne, varicose appearance. Some positive nerve ®bres were also observed accompanying blood vessels. These ®bres left nerve bundles and blood vessels, and surrounded NADPH-diaphorase-positive cells (Fig. 1B) or terminated as free nerve endings (Fig. 1C). Nearly 30% (137/505) of NADPHdiaphorase-positive cells were seen in close contact with the positive nerve ®bres. The double-immuno¯uorescence method in combination with NADPH-diaphorase histochemistry revealed coexpression of NADPH-diaphorase activity
H. Ichikawa, T. Sugimoto / Archives of Oral Biology 43 (1998) 167±171
169
Fig. 1. Photomicrographs of NADPH-diaphorase activity (A±D, G), inducible nitric oxide synthase (iNOS) immunoreactivity (E, I, J), S100-immunoreactivity (F) and neuronal nitric oxide synthase (nNOS) immunoreactivity (H) in the lingual portion of the incisor periodontal ligament. NADPH-diaphorase-positive cells have various shapes with or without processes (arrows in A, B). NADPHdiaphorase-positive nerve ®bres surround the positive cells (arrowheads in B) in close apposition to a blood vessel (bv in B) or terminate as free nerve endings (arrowheads in C). (D±F) and (G, H) are the same ®elds of views. NADPH-diaphorase-positive cells (arrows in D) coexpress both iNOS (arrows in E) and S100 immunoreactivities (arrows in F). The distribution of NADPH-diaphorase-positive nerve ®bres (arrowheads in G) is very similar to that of nNOS-immunoreactive nerve ®bres (arrowheads in H). Arrows in (G) and (H) indicate NADPH-diaphorase-positive cells (G) which are devoid of nNOS immunoreactivity (H). (I) and (J) show electron micrographs of a Runi-like ending obtained from adjacent sections. A terminal Schwann cell surrounding a Runi-like ending (s in I) contains dense reaction products for iNOS immunoreactivity (large arrowheads in I) and is covered with multiple layers of basal lamina (small arrows in I). Large arrows in (I) and (J) indicate the same short projection of the axoplasm. Clusters of small vesicles are seen at the base of the projection (small arrowheads in I and J). Scale bars: 50 mm (A±C), 100 mm (D±H) and 1 mm (I, J).
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with immunoreactivities for inducible and neuronal nitric oxide synthase and S100 in the periodontal ligament. All NADPH-diaphorase-positive cells coexpressed the inducible synthase and S100 immunoreactivities. In addition, all cells immunoreactive for inducible nitric oxide synthase coexpressed NADPH-diaphorase activity and S100 immunoreactivity (Fig. 1D±F). In nerve bundles, however, S100-immunoreactive Schwann cells were devoid of NADPHdiaphorase activity and inducible synthase immunoreactivity. NADPH-diaphorase-positive cells were always devoid of immunoreactivity for neuronal nitric oxide synthase. The distributions of NADPH-diaphorase-positive nerve ®bres and neuronal nitric oxide synthase-immunoreactive ones were identical (Fig. 1, G, H). No nerve ®bres immunoreactive for the inducible synthase were observed in the periodontal ligament. The coexpression study demonstrated that NADPHdiaphorase-positive cells and cells immunoreactive for inducible nitric oxide synthase had an identical distribution in the periodontal ligament of incisor teeth. Thus, inducible synthase-immunoreactive cells were examined by an immunoelectron-microscopic method to characterize NADPH-diaphorase-positive cells. The clusters of inducible synthase-immunoreactive cells in the periodontal ligament turned out to be Runi-like endings (Fig. 1, I). Terminal Schwann cells surrounding Runi-like endings contained numerous pinocytotic vesicles and some mitochondria, and formed lamellar sheets around the axoplasm. These Schwann cells were covered with multiple layers of basal lamina. The axoplasm was enriched with mitochondria (Fig. 1, I) and formed short projections that extended into the surrounding intercellular space (Fig. 1, J). Clusters of small vesicles were often seen at the base of the projections. It was the Schwann cell and its process that expressed immunoreactivity for inducible nitric oxide synthase in the Runi-like endings, while the axoplasm was devoid of it. At a higher magni®cation, the immunoreaction products were distributed over membranes of pinocytotic vesicles and the cytoplasm in Schwann cells. Neural NADPH-diaphorase activity could not be observed in the tissues covering the labial portion of the roots of incisor teeth. We demonstrate that the periodontal ligament of rat incisor teeth contains neural NADPH-diaphorase activity. NADPH-diaphorase-positive cells that coexpressed immunoreactivities for inducible nitric oxide synthase and S100 were distributed in the lingual portion of the ligament. Our electron-microscopic analysis for the inducible synthase immunoreactivity indicated that these NADPH-diaphorase-positive cells were identical to Schwann cells and their processes associated with Runi-like endings. This is supported by previous
®ndings that glial cells in the brainstem contained NADPH-diaphorase activity and immunoreactivity for inducible nitric oxide synthase (Galea et al., 1992; Paakkari and Lindsberg, 1995) and that Schwann cells in periodontal Runi-like endings exhibited S100 immunoreactivity (Sato et al., 1988). Because Schwann cells in nerve bundles were devoid of NADPH-diaphorase activity and the inducible synthase immunoreactivity, these cells are probably unable to synthesize nitric oxide. Thus, nitric oxide may be associated with the functions of Schwann cells speci®c to Runi-like endings surrounding the incisor periodontal ligament. We also demonstrate that nerve ®bres in the incisor periodontal ligament coexpress NADPH-diaphorase activity and immunoreactivity for neuronal nitric oxide synthase. Because both primary sensory neurones in the trigeminal ganglion and parasympathetic postganglionic neurones in the otic and pterygopalatine ganglia contain NADPH-diaphorase activity (Aimi et al., 1991; Alm et al., 1995; Morris et al., 1993; Kerezoudis et al., 1993b), the origin of these positively stained periodontal nerve ®bres is still unclear. However, their sensory nature is probably suggested by the distribution of their terminals; NADPH-diaphorase-positive nerve ®bres terminated as free nerve endings or were in contact with Schwann cells in Runi-like endings. This may be supported by our control ®ndings that virtually all NADPH-diaphorasepositive nerve ®bres in the incisor periodontal ligament disappeared after transection of the inferior alveolar nerve. The possibility that these nerve ®bres originate from the trigeminal mesencephalic nucleus is excluded, because primary sensory neurones in that nucleus are devoid of the enzyme activity (Vincent and Kimura, 1992) and because periodontal receptors from that nucleus other than Runi-like endings have not been reported (Byers et al., 1986). Thus, it can be deduced that NADPH-diaphorase-positive nerve ®bres in the periodontal ligament at least partly originate from the trigeminal ganglion. The coexpression of CGRP and substance P immunoreactivities by the NADPH-diaphorase-positive trigeminal cells (Aimi et al., 1991) and the demonstrated free nerve endings strongly suggest that at least some of the NADPH-diaphorase-positive trigeminal neurones are involved in nociception. It is possible that nitric oxide synthesized by trigeminal neurones is involved in sensory signal transduction (Paakkari and Lindsberg, 1995). On the other hand, the preferential distribution of NADPH-diaphorase activity and nitric oxide synthase immunoreactivity in the periodontal ligament of the incisor but not molar teeth may suggest that the neural nitric oxide is related to the speci®c tissue environment. Because the adult rodent incisor is continually erupting, the sensory endings in its periodontal ligament have to accommodate more
H. Ichikawa, T. Sugimoto / Archives of Oral Biology 43 (1998) 167±171
tissue reorganization than is the case for molars. Nitric oxide may thus be involved in the plasticity of sensory nerve endings in the rapidly erupting incisor periodontal ligament.
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