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 Experimental Research Department and Second Institute of Physiology, Semmelweis University of Medicine, U ¨ llo ˝i u ´t 78/A, 1082 Budapest, Hungary 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; Infer- ior alveolar nerve; Inferior alveolar artery; Dental pulp; Gingiva; Cat; Dog There is a good experimental evidence that the free radi- cal 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 exten- sively. Nicotinamide adenine dinucleotide phosphate-dia- phorase (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 con- trol 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 immunocytochemistry in the dental pulp, gingiva and infer- ior 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 sal- ine, 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 dis- sected 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 nicoti- namide 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 con- trols, where NADPH was omitted no reactivity was observed. Adjacent sections were incubated for 24 h at Neuroscience Letters 227 (1997) 91–94 0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(97)00319-4 * Corresponding author. Fax: +36 1 3343162; e-mail: lohinai@elet2.sote.hu