- Email: [email protected]
Nitric oxide synthase containing nerves in the cat and dog dental pulp and gingiva
Neuroscience Letters 227 (1997) 91–94
Nitric oxide synthase containing nerves in the cat and dog dental pulp and gingiva Zsolt Lohinai a ,*, Andrea D. Sze´kely b, Pe´ter Benedek c, Andra´s Csillag b a
¨ llo˝i u´t 78/A, 1082 Budapest, Hungary Experimental Research Department and Second Institute of Physiology, Semmelweis University of Medicine, U b Department of Anatomy, Semmelweis University of Medicine, Tu˝zolto´ utca 58, 1095 Budapest, Hungary c Department of Pathophysiology, Semmelweis University of Medicine, Nagyva´rad te´r 4, 1089 Budapest, Hungary Received 24 March 1997; revised version received 21 April 1997; accepted 22 April 1997
Abstract In a previous study we found that nitric oxide (NO) plays an essential role in the hemodynamic regulation of the feline dental pulp. However, no evidence for the presence of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) containing nerve fibers was found in the rat and cat dental pulps. In the present study, we are first to report the presence of a small number of NADPH-d positive and/or NO synthase immunoreactive perivascular and solitary varicose axons in the dental pulp and abundant number of similar axons in the gingiva of cats and dogs. These fibres may travel within the inferior alveolar nerve and might participate in sensory (i.e. pain) as well as in autonomic (i.e. regulation of blood flow) innervation of the dental pulp and gingiva. 1997 Elsevier Science Ireland Ltd. Keywords: Nitric oxide synthase; Nicotinamide adenine dinucleotide phosphate-diaphorase; Histochemistry; Immunocytochemistry; Inferior alveolar nerve; Inferior alveolar artery; Dental pulp; Gingiva; Cat; Dog
There is a good experimental evidence that the free radical nitric oxide (NO) plays an important role as a neuronal messenger molecule in the digestive tract [16]. NO synthase (NOS) immunoreactivity is localized within neurons and their processes throughout the rat gastrointestinal system [2]. The production and role of NO in the various structures of the oral cavity, however, has not been investigated extensively. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d; one possible histochemical marker for NOS [7]) positive neuronal elements were observed in mouse tongue [6] but no evidence for NADPH-d containing nerve fibers was found in the dental pulp, periodontium and alveolar bone of rat [8]. On the other hand, physiological data have demonstrated an essential role for NO in the control of vascular conductance in rat [9] and cat [11] dental pulp and rat gingiva [5], therefore, the aim of the present study was to examine the localization of NOS containing neural elements using NADPH-d histochemistry and NOS
* Corresponding author. Fax: +36 1 3343162; e-mail: [email protected]
immunocytochemistry in the dental pulp, gingiva and inferior alveolar nerve (IAN) of cat and dog. Six adult cats of either sex (body weight 2–2.5 kg) were terminally anaesthetized with intraperitoneal injections of sodium pentobarbitone and perfused via the aorta with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Following perfusion, gingiva (next to the canines), dental pulp (from extracted upper and lower canines), IAN and the trigeminal ganglion (TG) were dissected out, blocked and postfixed by immersion for 2 h. The blocks were then cryoprotected with 20% sucrose in PB at 4°C overnight and serial sections were cut on a freezing microtome at 30–60 mm. Following several rinses in PB, the sections were reacted in the presence of reduced nicotinamide adenine dinucleotide phosphate (b-NADPH; Sigma Ltd., Hungary) and nitro blue tetrazolium (NBT; Sigma Ltd., Hungary) according to Lohinai et al. [12]. The reaction was stopped when dark blue neurons/nervous elements appeared in the tissue. Then the sections were mounted on glass slides, dehydrated and coverslipped in DePeX. In controls, where NADPH was omitted no reactivity was observed. Adjacent sections were incubated for 24 h at
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0319-4
92
Z. Lohinai et al. / Neuroscience Letters 227 (1997) 91–94
4°C in a rabbit anti-NOS primary serum (Transduction Labs./Affiniti Research, UK; 1:1000 in 0.01 M phosphate buffered saline (PBS) pH 7.4), supposed to be specific for neuronal NOS [3]. Following rinses in PBS, the sections were reacted with a biotinylated goat-anti rabbit secondary serum (Vector Labs., Peterborough, UK; 1:100), then with the avidin-biotin complex (ABC Vectakit Elite; Vector Labs., Peterborough, UK; 1:100). The immunocomplex was visualized with 3,3,-diaminobenzidine (Sigma KFT, Budapest, Hungary). The sections were then mounted, dehydrated and covered in DePeX. In control sections, where the primary or secondary antibodies were replaced with normal goat serum, no immunostaining appeared. Gingiva, dental pulp (from upper and lower canines) and IAN were also dissected post-mortem from six dogs (body weight 15–36 kg) used for physiological experiments and fixed by immersion in 4% paraformaldehyde/0.1 M PB (pH 7.4) for approximately a week at 4°C. The further procedure was identical to that described above. In the feline dental pulp we found a small number of NADPH-d positive (NADPH-d+) nerve fibers. The reactive nerve fibres were scattered in the connective tissue framework of dental pulp, with the axons occupying a perivascular location rather than traversing the matrix following either straight or wavy courses. In longitudinal sections, the course of the slightly varicose fibres could be followed along the vessels with no obvious branchings or bifurcations (Fig. 1b,c). Similar distribution of fibres was observed both in cats and dogs and also, following NOS immunocytochemistry (Fig. 1a,d). In both species, the endothelial cells were reactive to NADPH-d. A rich network of profusely arborizing NADPH-d reactive axons was found in the subepithelial connective tissue (lamina propria) of gingiva, some of which surrounded vessels (Fig. 1e). The fibres appeared to follow the course of vessels and were also found in the vicinity of the epithelial layer of mucosa. Similar staining pattern of nerve fibers was achieved when using NOS immunocytochemistry. In both species, following NADPH-d histochemistry a dark blue, although diffuse, labelling appeared throughout the epithelium and a diffuse, light blue reaction product was found in the cytoplasm of endothelial cells. Occasionally, NADPHd + macrophages and mast cells were noted in the connective tissue (not shown). In both species studied, numerous axons stained with NADPH-d histochemistry or NOS immunocytochemistry were found within the fascicles of the IAN (Fig. 1f). In the vicinity of the inferior alveolar artery (IAA) some reac-
tive and slightly varicose fibres were also observed to follow the course of the artery (Fig. 1g). Similar to the previously described tissues, the endothelial layer of blood vessels showed NADPH-d reactivity. A considerable number of NADPH-d/NOS labelled small sized ganglionic cells and fibres were found in cat TG (Fig. 1h). The results of the current study confirmed that in the feline dental pulp, the endothelial cells are NADPH-d+ [8]. In addition, we found a small number of NADPH-d+ nerve fibers in cat and dog dental pulp, such NADPH-d+ elements have not been described yet. These observations are in contrast to results of Kerezoudis et al. [8], who failed to detect NADPH-d+ nerve fibers in rat and cat dental pulp. This difference might reflect interspecies differences, as is the case with the rat dental pulp where no vasoactive intestinal polypeptide (VIP) nerves are present as opposed to the dental pulp of other mammalian species [19]. Alternatively the use of different methods (e.g. we did not decalcify the tissue) may explain the apparent discrepancy between the present and previous studies. An NO dependent basal vasodilator tone is reported in rat [9] and cat [11] dental pulp. Presumably, under resting conditions the capacity of the NADPH-d+ endothelium to generate NO is more important than the small number of NADPH-d+/NOS+ nerves. Since the vascular conductance of cat dental pulp is highly responsive to exogenous NO [11], it may be speculated that the stimulated release of endogenous NO from endothelium and/or nitrergic nerves may also be involved in the control of the dental pulp vasculature. In rat incisor pulp a partial contribution of NO was found in carbachol-induced vasodilation [17], however, there was no evidence for an involvement of NO in neurogenic extravasation [10]. In rat gingiva, local capsaicin administration is followed by an increase in vascular conductance, a response in which NO is thought to play an important role [5]. However, NO does not seem to contribute to neurogenic extravasation [10]. Our results indicated that NO, released from perivascular nerves, endothelium and epithelium, may regulate local blood circulation. As in rat [8], the endothelial layer of blood vessels showed NADPH-d positivity in cat and dog. In the latter species, NADPH-d reactivity was observed throughout the epithelium, whereas in rats, solely the basal cell layer of the epithelium was reported to be NADPH-d+ [8]. These oral epithelial cells may have the capability to form NO, similar to the cells of the paranasal sinuses and nasal epithelium of human [13]. It is conceivable that such
Fig. 1. NOS and NADPH-d positive varicose fibres in the feline and canine oral tissues. (a) NOS immunoreactive fibres (arrowheads) in cat radicular pulp. (b) A parallel section of cat radicular pulp showing NADPH-d+ fibres (arrowheads). (c) NADPH-d+ fibres (arrowheads) in cat coronal dental pulp. (d) NOSpositive axons in dog coronal dental pulp. (e) NADPH-d reactive fibre bundles (arrowheads) in cat gingiva. Note the high level of diffuse reactivity in epithelial cells (arrows point at the epithelium-connective tissue interface). (f) NADPH-d reactive axons (arrowheads) in cat inferior alveolar nerve. (g) NOS immunopositive nerve fibres (arrowheads) in the vicinity of dog inferior alveolar artery. (h) NADPH-d labelled small sensory neurones in cat trigeminal ganglion. Scale bars, 50 mm.
Z. Lohinai et al. / Neuroscience Letters 227 (1997) 91–94
mechanism may contribute to the ‘aerocrine’ regulation of pulmonary function [13], help to protect against infectious agents and may control local blood flow as well.
93
Our study is the first in the literature which describes the occurrence of NADPH-d/NOS positive fibres in IAN. The presence of NOS containing perivascular fibres and
94
Z. Lohinai et al. / Neuroscience Letters 227 (1997) 91–94
NADPH-d reactive endothelium in IAA raises the possibility, that IAA is a potential site of vasoregulation of the supplied tissues rather than a mere vascular conduit in bone. Our results confirmed that in TG there is a considerable number of small sized NADPH-d/NOS labelled ganglionic cells [1]. According to Aimi et al. [1] many NADPH-d+ cells in rat TG also showed substance P and calcitonin gene-related peptide immunoreactivity, which are characteristic markers of sensory axons. Therefore, NOS containing sensory neurons of the TG may innervate dental pulp and gingiva via IAN. In rat [4] and pig [15] superior cervical ganglion a dense plexus of preganglionic fibers and minor portion of sympathetic ganglionic cells were NOS positive. These cells colocalize with VIP [15] and may innervate gingiva, but possibly not dental pulp, since VIP fibres in dental pulp do not disappear after sympathectomy [20]. The occurrence of parasympathetic nerves in dental pulp, as well as in gingiva, is still a matter of debate. The detection of VIP immunoreactive fibers in mammalian dental pulp [19] and human gingiva [14] lends support to the hypothesis that teeth and gingiva may also be supplied by parasympathetic nerves. The stimulation of rat vagal nerve evoked NO and VIP release [18] and similarly, a coexistence of NOS and VIP was verified in the sphenopalatine and submandibular ganglia of rat [4]. Therefore, the NOS containing fibres of dental pulp and gingiva are likely to colocalize with VIP, thus raising the possibility of their parasympathetic nature. This hypothesis requires further investigation. In conclusion, our study demonstrated the presence of NADPH-d+/NOS immunoreactive perivascular and solitary varicose axons in the dental pulp and gingiva of cats and dogs. It is conceivable that NO produced from these structures plays a role in either the sensory and/or autonomic innervation of oral tissues, including IAN-IAA, gingiva and dental pulp, potentially subserving mechanisms such as pain perception, control of blood flow or modulation of inflammation. Epithelial cells of gingiva by NO production may contribute to the ‘aerocrine’ regulation of pulmonary function and may protect against infectious agents.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17]
This work was supported by grants of the Hungarian National Research Funds OTKA No. F-20469 and ETK I13. [1] Aimi, Y., Fujimura, M., Vincent, S.R. and Kimura, H., Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat, J. Comp. Neurol., 306 (1991) 382–392. [2] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neuronal role for nitric oxide, Nature, 347 (1990) 768–770. [3] Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed, R.R.
[18]
[19]
[20]
and Snyder, S.H., Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature, 351 (1991) 714–718. Ceccatelli, S., Lundberg, J.B., Zhang, X., Aman, K. and Ho¨kfelt, T., Immunohistochemical demonstration of nitric oxide synthase in the peripheral autonomic nervous system, Brain Res., 656 (1994) 381– 395. ´ ., Matheny, J.L., Roth, G.I. and Richardson, D.R., Effect Fazekas, A of nitric oxide inhibition on capsaicin-elicited vasodilation in the rat oral circulation, Res. Exp. Med., 194 (1994) 357–365. Grozdanovic, Z., Baumgarten, H.G. and Bru¨ning, G., Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse, Neuroscience, 48 (1992) 225–239. Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH-diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811–2814. Kerezoudis, N.P., Olgart, L. and Fried, K., Localization of NADPHdiaphorase activity in the dental pulp, perodontium and alveolar bone of the rat, Histochemistry, 100 (1993) 319–322. Kerezoudis, N.P., Olgart, L. and Edwall, L., Differential effects of nitric oxide synthesis inhibition on basal blood flow and antidromic vasodilation in rat oral tissues, Eur. J. Pharmacol., 241 (1993) 209– 219. Kerezoudis, N.P., Olgart, L. and Edwall, L., Involvement of substance P but not nitric oxide or calcitonin gene-related peptide in neurogenic plasma extravasation in rat incisor pulp and lip, Arch. Oral Biol., 39 (1994) 769–774. Lohinai, Z., Balla, I., Marczis, J., Vass, Z. and Kova´ch, A.G.B., Evidence for the role of nitric oxide in the circulation of the dental pulp, J. Dent. Res., 74 (1995) 1501–1506. Lohinai, Zs., Sze´kely, A.D., Soo´s, L. and Fehe´r, E., Distribution of nitric oxide synthase containing elements in the feline submandibular gland, Neurosci. Lett., 192 (1995) 9–12. Lundberg, J., Airborne nitric oxide: inflammatory marker and aerocrine messenger in man, Acta Physiol. Scand., 157 (S633 )(1996) 1– 27. Luthman, J., Johansson, O., Ahlstro¨m, U. and Kvint, S., Immunohistochemical studies of the neurochemical markers, CGRP, enkephalin, galanin, y-MSH, NPY, PHI, proctolin, PTH, somatostatin, SP, VIP, tyrosinehydroxylase and neurofilament in nerves and cells of the human attached gingiva, Arch. Oral Biol., 33 (1988) 149–158. Modin, A., Weitzberg, E., Ho¨kfelt, T. and Lundberg, J.M., Nitric oxide synthase in the pig autonomic nervous system in relation to the influence of N-nitro-l-arginine on sympathetic and parasympathetic vascular control in vivo, Neuroscience, 62 (1994) 189–203. Moncada, S., The 1991 Ulf von Euler lecture. The l-arginine: nitric oxide pathway, Acta Physiol. Scand., 145 (1992) 201–227. Olgart, L., Kostouros, G.D. and Edwall, L., Local actions of acethylcholine on vasomotor regulation in rat incisor pulp, Acta Physiol. Scand., 158 (1996) 311–316. Takahashi, T. and Owyang, C., Vagal control of nitric oxide and vasoactive intestinal polypeptide release in the regulation of gastric relaxation in rat, J. Physiol. Lond., 484 (1995) 481–492. Uddman, R., Bjo¨rlin, G., Mo¨ller, B. and Sundler, F., Occurrence of VIP nerves in mammalian dental pulps, Acta Odontol. Scand., 38 (1980) 325–328. Wakisaka, S., Ichikawa, H. and Akai, M.J., The distribution and origins of peptide- and catecholamine-containing nerve fibres in the feline dental pulp and effects of cavity preparation on these nerve fibres, J. Osaka Univ. Dent. Sch., 26 (1986) 17–28.
Nitric oxide synthase containing nerves in the cat and dog dental pulp and gingiva Zsolt Lohinai a ,*, Andrea D. Sze´kely b, Pe´ter Benedek c, Andra´s Csillag b a
¨ llo˝i u´t 78/A, 1082 Budapest, Hungary Experimental Research Department and Second Institute of Physiology, Semmelweis University of Medicine, U b Department of Anatomy, Semmelweis University of Medicine, Tu˝zolto´ utca 58, 1095 Budapest, Hungary c Department of Pathophysiology, Semmelweis University of Medicine, Nagyva´rad te´r 4, 1089 Budapest, Hungary Received 24 March 1997; revised version received 21 April 1997; accepted 22 April 1997
Abstract In a previous study we found that nitric oxide (NO) plays an essential role in the hemodynamic regulation of the feline dental pulp. However, no evidence for the presence of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) containing nerve fibers was found in the rat and cat dental pulps. In the present study, we are first to report the presence of a small number of NADPH-d positive and/or NO synthase immunoreactive perivascular and solitary varicose axons in the dental pulp and abundant number of similar axons in the gingiva of cats and dogs. These fibres may travel within the inferior alveolar nerve and might participate in sensory (i.e. pain) as well as in autonomic (i.e. regulation of blood flow) innervation of the dental pulp and gingiva. 1997 Elsevier Science Ireland Ltd. Keywords: Nitric oxide synthase; Nicotinamide adenine dinucleotide phosphate-diaphorase; Histochemistry; Immunocytochemistry; Inferior alveolar nerve; Inferior alveolar artery; Dental pulp; Gingiva; Cat; Dog
There is a good experimental evidence that the free radical nitric oxide (NO) plays an important role as a neuronal messenger molecule in the digestive tract [16]. NO synthase (NOS) immunoreactivity is localized within neurons and their processes throughout the rat gastrointestinal system [2]. The production and role of NO in the various structures of the oral cavity, however, has not been investigated extensively. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d; one possible histochemical marker for NOS [7]) positive neuronal elements were observed in mouse tongue [6] but no evidence for NADPH-d containing nerve fibers was found in the dental pulp, periodontium and alveolar bone of rat [8]. On the other hand, physiological data have demonstrated an essential role for NO in the control of vascular conductance in rat [9] and cat [11] dental pulp and rat gingiva [5], therefore, the aim of the present study was to examine the localization of NOS containing neural elements using NADPH-d histochemistry and NOS
* Corresponding author. Fax: +36 1 3343162; e-mail: [email protected]
immunocytochemistry in the dental pulp, gingiva and inferior alveolar nerve (IAN) of cat and dog. Six adult cats of either sex (body weight 2–2.5 kg) were terminally anaesthetized with intraperitoneal injections of sodium pentobarbitone and perfused via the aorta with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Following perfusion, gingiva (next to the canines), dental pulp (from extracted upper and lower canines), IAN and the trigeminal ganglion (TG) were dissected out, blocked and postfixed by immersion for 2 h. The blocks were then cryoprotected with 20% sucrose in PB at 4°C overnight and serial sections were cut on a freezing microtome at 30–60 mm. Following several rinses in PB, the sections were reacted in the presence of reduced nicotinamide adenine dinucleotide phosphate (b-NADPH; Sigma Ltd., Hungary) and nitro blue tetrazolium (NBT; Sigma Ltd., Hungary) according to Lohinai et al. [12]. The reaction was stopped when dark blue neurons/nervous elements appeared in the tissue. Then the sections were mounted on glass slides, dehydrated and coverslipped in DePeX. In controls, where NADPH was omitted no reactivity was observed. Adjacent sections were incubated for 24 h at
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0319-4
92
Z. Lohinai et al. / Neuroscience Letters 227 (1997) 91–94
4°C in a rabbit anti-NOS primary serum (Transduction Labs./Affiniti Research, UK; 1:1000 in 0.01 M phosphate buffered saline (PBS) pH 7.4), supposed to be specific for neuronal NOS [3]. Following rinses in PBS, the sections were reacted with a biotinylated goat-anti rabbit secondary serum (Vector Labs., Peterborough, UK; 1:100), then with the avidin-biotin complex (ABC Vectakit Elite; Vector Labs., Peterborough, UK; 1:100). The immunocomplex was visualized with 3,3,-diaminobenzidine (Sigma KFT, Budapest, Hungary). The sections were then mounted, dehydrated and covered in DePeX. In control sections, where the primary or secondary antibodies were replaced with normal goat serum, no immunostaining appeared. Gingiva, dental pulp (from upper and lower canines) and IAN were also dissected post-mortem from six dogs (body weight 15–36 kg) used for physiological experiments and fixed by immersion in 4% paraformaldehyde/0.1 M PB (pH 7.4) for approximately a week at 4°C. The further procedure was identical to that described above. In the feline dental pulp we found a small number of NADPH-d positive (NADPH-d+) nerve fibers. The reactive nerve fibres were scattered in the connective tissue framework of dental pulp, with the axons occupying a perivascular location rather than traversing the matrix following either straight or wavy courses. In longitudinal sections, the course of the slightly varicose fibres could be followed along the vessels with no obvious branchings or bifurcations (Fig. 1b,c). Similar distribution of fibres was observed both in cats and dogs and also, following NOS immunocytochemistry (Fig. 1a,d). In both species, the endothelial cells were reactive to NADPH-d. A rich network of profusely arborizing NADPH-d reactive axons was found in the subepithelial connective tissue (lamina propria) of gingiva, some of which surrounded vessels (Fig. 1e). The fibres appeared to follow the course of vessels and were also found in the vicinity of the epithelial layer of mucosa. Similar staining pattern of nerve fibers was achieved when using NOS immunocytochemistry. In both species, following NADPH-d histochemistry a dark blue, although diffuse, labelling appeared throughout the epithelium and a diffuse, light blue reaction product was found in the cytoplasm of endothelial cells. Occasionally, NADPHd + macrophages and mast cells were noted in the connective tissue (not shown). In both species studied, numerous axons stained with NADPH-d histochemistry or NOS immunocytochemistry were found within the fascicles of the IAN (Fig. 1f). In the vicinity of the inferior alveolar artery (IAA) some reac-
tive and slightly varicose fibres were also observed to follow the course of the artery (Fig. 1g). Similar to the previously described tissues, the endothelial layer of blood vessels showed NADPH-d reactivity. A considerable number of NADPH-d/NOS labelled small sized ganglionic cells and fibres were found in cat TG (Fig. 1h). The results of the current study confirmed that in the feline dental pulp, the endothelial cells are NADPH-d+ [8]. In addition, we found a small number of NADPH-d+ nerve fibers in cat and dog dental pulp, such NADPH-d+ elements have not been described yet. These observations are in contrast to results of Kerezoudis et al. [8], who failed to detect NADPH-d+ nerve fibers in rat and cat dental pulp. This difference might reflect interspecies differences, as is the case with the rat dental pulp where no vasoactive intestinal polypeptide (VIP) nerves are present as opposed to the dental pulp of other mammalian species [19]. Alternatively the use of different methods (e.g. we did not decalcify the tissue) may explain the apparent discrepancy between the present and previous studies. An NO dependent basal vasodilator tone is reported in rat [9] and cat [11] dental pulp. Presumably, under resting conditions the capacity of the NADPH-d+ endothelium to generate NO is more important than the small number of NADPH-d+/NOS+ nerves. Since the vascular conductance of cat dental pulp is highly responsive to exogenous NO [11], it may be speculated that the stimulated release of endogenous NO from endothelium and/or nitrergic nerves may also be involved in the control of the dental pulp vasculature. In rat incisor pulp a partial contribution of NO was found in carbachol-induced vasodilation [17], however, there was no evidence for an involvement of NO in neurogenic extravasation [10]. In rat gingiva, local capsaicin administration is followed by an increase in vascular conductance, a response in which NO is thought to play an important role [5]. However, NO does not seem to contribute to neurogenic extravasation [10]. Our results indicated that NO, released from perivascular nerves, endothelium and epithelium, may regulate local blood circulation. As in rat [8], the endothelial layer of blood vessels showed NADPH-d positivity in cat and dog. In the latter species, NADPH-d reactivity was observed throughout the epithelium, whereas in rats, solely the basal cell layer of the epithelium was reported to be NADPH-d+ [8]. These oral epithelial cells may have the capability to form NO, similar to the cells of the paranasal sinuses and nasal epithelium of human [13]. It is conceivable that such
Fig. 1. NOS and NADPH-d positive varicose fibres in the feline and canine oral tissues. (a) NOS immunoreactive fibres (arrowheads) in cat radicular pulp. (b) A parallel section of cat radicular pulp showing NADPH-d+ fibres (arrowheads). (c) NADPH-d+ fibres (arrowheads) in cat coronal dental pulp. (d) NOSpositive axons in dog coronal dental pulp. (e) NADPH-d reactive fibre bundles (arrowheads) in cat gingiva. Note the high level of diffuse reactivity in epithelial cells (arrows point at the epithelium-connective tissue interface). (f) NADPH-d reactive axons (arrowheads) in cat inferior alveolar nerve. (g) NOS immunopositive nerve fibres (arrowheads) in the vicinity of dog inferior alveolar artery. (h) NADPH-d labelled small sensory neurones in cat trigeminal ganglion. Scale bars, 50 mm.
Z. Lohinai et al. / Neuroscience Letters 227 (1997) 91–94
mechanism may contribute to the ‘aerocrine’ regulation of pulmonary function [13], help to protect against infectious agents and may control local blood flow as well.
93
Our study is the first in the literature which describes the occurrence of NADPH-d/NOS positive fibres in IAN. The presence of NOS containing perivascular fibres and
94
Z. Lohinai et al. / Neuroscience Letters 227 (1997) 91–94
NADPH-d reactive endothelium in IAA raises the possibility, that IAA is a potential site of vasoregulation of the supplied tissues rather than a mere vascular conduit in bone. Our results confirmed that in TG there is a considerable number of small sized NADPH-d/NOS labelled ganglionic cells [1]. According to Aimi et al. [1] many NADPH-d+ cells in rat TG also showed substance P and calcitonin gene-related peptide immunoreactivity, which are characteristic markers of sensory axons. Therefore, NOS containing sensory neurons of the TG may innervate dental pulp and gingiva via IAN. In rat [4] and pig [15] superior cervical ganglion a dense plexus of preganglionic fibers and minor portion of sympathetic ganglionic cells were NOS positive. These cells colocalize with VIP [15] and may innervate gingiva, but possibly not dental pulp, since VIP fibres in dental pulp do not disappear after sympathectomy [20]. The occurrence of parasympathetic nerves in dental pulp, as well as in gingiva, is still a matter of debate. The detection of VIP immunoreactive fibers in mammalian dental pulp [19] and human gingiva [14] lends support to the hypothesis that teeth and gingiva may also be supplied by parasympathetic nerves. The stimulation of rat vagal nerve evoked NO and VIP release [18] and similarly, a coexistence of NOS and VIP was verified in the sphenopalatine and submandibular ganglia of rat [4]. Therefore, the NOS containing fibres of dental pulp and gingiva are likely to colocalize with VIP, thus raising the possibility of their parasympathetic nature. This hypothesis requires further investigation. In conclusion, our study demonstrated the presence of NADPH-d+/NOS immunoreactive perivascular and solitary varicose axons in the dental pulp and gingiva of cats and dogs. It is conceivable that NO produced from these structures plays a role in either the sensory and/or autonomic innervation of oral tissues, including IAN-IAA, gingiva and dental pulp, potentially subserving mechanisms such as pain perception, control of blood flow or modulation of inflammation. Epithelial cells of gingiva by NO production may contribute to the ‘aerocrine’ regulation of pulmonary function and may protect against infectious agents.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17]
This work was supported by grants of the Hungarian National Research Funds OTKA No. F-20469 and ETK I13. [1] Aimi, Y., Fujimura, M., Vincent, S.R. and Kimura, H., Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat, J. Comp. Neurol., 306 (1991) 382–392. [2] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neuronal role for nitric oxide, Nature, 347 (1990) 768–770. [3] Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed, R.R.
[18]
[19]
[20]
and Snyder, S.H., Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature, 351 (1991) 714–718. Ceccatelli, S., Lundberg, J.B., Zhang, X., Aman, K. and Ho¨kfelt, T., Immunohistochemical demonstration of nitric oxide synthase in the peripheral autonomic nervous system, Brain Res., 656 (1994) 381– 395. ´ ., Matheny, J.L., Roth, G.I. and Richardson, D.R., Effect Fazekas, A of nitric oxide inhibition on capsaicin-elicited vasodilation in the rat oral circulation, Res. Exp. Med., 194 (1994) 357–365. Grozdanovic, Z., Baumgarten, H.G. and Bru¨ning, G., Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse, Neuroscience, 48 (1992) 225–239. Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH-diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811–2814. Kerezoudis, N.P., Olgart, L. and Fried, K., Localization of NADPHdiaphorase activity in the dental pulp, perodontium and alveolar bone of the rat, Histochemistry, 100 (1993) 319–322. Kerezoudis, N.P., Olgart, L. and Edwall, L., Differential effects of nitric oxide synthesis inhibition on basal blood flow and antidromic vasodilation in rat oral tissues, Eur. J. Pharmacol., 241 (1993) 209– 219. Kerezoudis, N.P., Olgart, L. and Edwall, L., Involvement of substance P but not nitric oxide or calcitonin gene-related peptide in neurogenic plasma extravasation in rat incisor pulp and lip, Arch. Oral Biol., 39 (1994) 769–774. Lohinai, Z., Balla, I., Marczis, J., Vass, Z. and Kova´ch, A.G.B., Evidence for the role of nitric oxide in the circulation of the dental pulp, J. Dent. Res., 74 (1995) 1501–1506. Lohinai, Zs., Sze´kely, A.D., Soo´s, L. and Fehe´r, E., Distribution of nitric oxide synthase containing elements in the feline submandibular gland, Neurosci. Lett., 192 (1995) 9–12. Lundberg, J., Airborne nitric oxide: inflammatory marker and aerocrine messenger in man, Acta Physiol. Scand., 157 (S633 )(1996) 1– 27. Luthman, J., Johansson, O., Ahlstro¨m, U. and Kvint, S., Immunohistochemical studies of the neurochemical markers, CGRP, enkephalin, galanin, y-MSH, NPY, PHI, proctolin, PTH, somatostatin, SP, VIP, tyrosinehydroxylase and neurofilament in nerves and cells of the human attached gingiva, Arch. Oral Biol., 33 (1988) 149–158. Modin, A., Weitzberg, E., Ho¨kfelt, T. and Lundberg, J.M., Nitric oxide synthase in the pig autonomic nervous system in relation to the influence of N-nitro-l-arginine on sympathetic and parasympathetic vascular control in vivo, Neuroscience, 62 (1994) 189–203. Moncada, S., The 1991 Ulf von Euler lecture. The l-arginine: nitric oxide pathway, Acta Physiol. Scand., 145 (1992) 201–227. Olgart, L., Kostouros, G.D. and Edwall, L., Local actions of acethylcholine on vasomotor regulation in rat incisor pulp, Acta Physiol. Scand., 158 (1996) 311–316. Takahashi, T. and Owyang, C., Vagal control of nitric oxide and vasoactive intestinal polypeptide release in the regulation of gastric relaxation in rat, J. Physiol. Lond., 484 (1995) 481–492. Uddman, R., Bjo¨rlin, G., Mo¨ller, B. and Sundler, F., Occurrence of VIP nerves in mammalian dental pulps, Acta Odontol. Scand., 38 (1980) 325–328. Wakisaka, S., Ichikawa, H. and Akai, M.J., The distribution and origins of peptide- and catecholamine-containing nerve fibres in the feline dental pulp and effects of cavity preparation on these nerve fibres, J. Osaka Univ. Dent. Sch., 26 (1986) 17–28.