1 Excessive production of nitric oxide (NO), and the generation of peroxynitrite have been implicated in various proin¯ammatory conditions. In the present study, using mercaptoethylguanidine (MEG), a selective inhibitor of iNOS and a peroxynitrite scavenger, we investigated the role of iNOS and peroxynitrite in a rat model of periodontitis. 2 Periodontitis was produced in rat by a ligature of 2/0 braided silk placed around the cervix of the lower left 1st molar. Animals were then divided into two groups: one group of rats was treated with MEG (30 mg kg 71 , i.p., 4 times per day for 8 days), animals in the other group received vehicle. At day 8, the gingivomucosal tissue encircling the mandibular 1st molars was removed on both sides from ligated and sham operated animals for inducible nitric oxide synthase (iNOS) activity assay and for immunocytochemistry with anti-iNOS serum. Plasma extravasation was measured with the Evans blue technique. Alveolar bone loss was measured with a videomicroscopy. 3 Ligation caused a signi®cant, more than 3 fold increase in the gingival iNOS activity, whereas it did not a ect iNOS activity on the contralateral side, when compared to sham-operated animals. Immunohistochemical analysis revealed iNOS-positive macrophages, lymphocytes and PMNs in the connective tissue and immunoreactive basal layers of epithelium on side of the ligature, and only a few iNOS-negative connective tissue cells on the contralateral side. Ligation signi®cantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction compared to the contralateral side. MEG treatment signi®cantly reduced the plasma extravasation and bone destruction. 4 The present results demonstrated that ligature-induced periodontitis increases local NO production and that MEG treatment protects against the associated extravasation and bone destruction. Based on the present data, we propose that enhanced formation of NO and peroxynitrite plays a signi®cant role in the pathogenesis of periodontitis.

Effect of ligation on the vascular permeability in the gingiva and alveolar mucosa encircling the mandibular left 1st molar was studied in rats with and without capsaicin pretreatment. Vascular permeability was assessed by the Evans blue extravasation. Ligation caused a significant augmentation in vascular permeability of the gingivomucosal tissue at day 8 (right: 18.14 +/- 1.68 micrograms g-1; left (ligature): 38.21 +/- 2.43 micrograms g-1, n = 8, p < 0.001) and at day 14 (right: 20.31 +/- 1.71 micrograms g-1: left (ligature): 36.98 +/- 2.73 micrograms g-1, n = 8, p < 0.001). 4 days after ligation, no difference could be observed in vascular permeability in the oral mucosa of the ligated side (left: 23.14 +/- 1.21 micrograms g-1) as compared to the side without ligature (right: 23.5 +/- 1.45 micrograms g-1, n = 8, NS). There was no elevation of vascular permeability of gingivomuscosal tissue around the ligation in rats pretreated with capsaicin either in newborn age (right: 23.92 +/- 1.76 micrograms g-1; left (ligature): 23.51 +/- 2.16 micrograms g-1, n = 8, NS) or in adult age (right: 20.61 +/- 1.62 micrograms g-1; left (ligature): 20.85 +/- 1.07 micrograms g-1, n = 8, NS). Light microscopical studies of oral mucosa revealed, that 8 and 14 days after the ligature placed around the mandibular left 1st molar of the rat, there resulted an accumulation of inflammatory cells in the connective tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Since the rôle of neurogenic inflammation in various pathological conditions is well-established, we presume that it also has great importance in the development of several inflammatory processes in the oral mucosa. In the course of experimentally-induced neurogenic inflammation of the oral mucosa in rats, vasodilation and plasma extravasation occur in the area supplied by unmyelinated capsaicin-sensitive fibres. Upon activating these fibres in the oral mucosa, mediators such as histamine, substance P (SP) and calcitonin gene related peptide (CGRP) are released from the peripheral terminals of afferent nerves causing characteristic symptoms of neurogenic inflammation. By histochemical techniques, SP- and CGRP-immunoreactive fibres have occurred in the area of the rat oral mucosa stimulated mostly in the free and attached gingiva around the molar teeth in the lower jaw. Capsaicin pretreatment performed neonatally or at adult age causes a loss of SP-immunoreactive fibres and prevents the vasodilatory responses, as well as the increase in vascular permeability elicited by the antidromic stimulation of the inferior alveolar nerve. As the transection of inferior alveolar nerve decreased the extravasation of Evans blue on the ipsilateral side and did not affect the capsaicin-induced enhancement in blood flow, we suggest that the two symptoms of the inflammation, i.e., the increased vascular permeability and decreased vascular resistance, should be produced by different mechanisms. The results of all the morphological and functional studies seem to confirm the possibility that there is an important neurogenic component of the inflammatory alterations caused by different mechanical and chemical stimuli in the oral mucosa.

There is no remarkable VEGF production under physiologic circumstances in rat gingiva, but VEGF is able to increase gingival blood flow through the activation of VEGF2 receptors. Furthermore, NO release may contribute to VEGF's vasodilatory effect.

In the present series of investigations we first studied the local effects of exogenous substance P (SP) on the hallmarks of neurogenic inflammation, i.e. vascular permeability and blood flow, in the oral mucosa of the rat. Pretreatment with capsaicin was shown to attenuate the symptoms of neurogenic inflammation; therefore, the distribution of nerve fibers displaying substance P-like immunoreactivity (SP-IR) in the mandibular mucosa was also assessed in control rats and in animals pretreated with capsaicin both neonatally and in adulthood using immunohistochemical techniques. The application of SP at a dose of 7.5 nmol resulted in an almost 70% increase of vascular permeability (NS) and the administration of a four-fold higher dose (30 nmol) produced about 150% increase in Evans blue extravasation compared with control values (p < 0.05). A similar increase (ca 146%) in vascular permeability was observed in response to 45 nmol SP solution (p < 0.05). While the 7.5 nmol SP-solution failed to affect blood flow, the 30 nmol SP significantly increased it by ca. 38% (p < 0.05). The administration of the 45 nmol SP solution resulted in a similar enhancement of blood flow (43%, p < 0.05). Capsaicin pretreatment performed either neonatally or in adulthood has reduced the number of SP-immunoreactive fibers in the oral mucosa. Our functional results suggest that SP may have a role in the experimentally-induced neurogenic inflammation of the oral mucosa in the rat. This is also supported by our finding that capsaicin pretreatment, known to decrease the number of SP-immunoreactive fibers in these tissues, reduced the symptoms of neurogenic inflammation.