The Expression of Nitric Oxide in the Gingival Tissue in Subjects with Periodontitis and Chronic Pain / Expression de Oxido Nítrico en el Tejido Gingival de Sujetos con Periodontitis y Dolor Crónico

Periodontal disease (PD) is a chronic infection that may have local and systemic rebound. Although a series of inflammatory mediators are involved in PD, the mechanisms involved in chronic craniofacial pain associated with it require elucidation. The aim of this study was to evaluate the immunoreactivity of substance P (SP), neuronal (nNOS) and inducible (iNOS) nitric oxide synthases in gingival samples of patients with severe PD with and without chronic craniofacial pain. Gingival specimens were obtained during routine periodontal surgery while managing 20 patients with both PD and chronic craniofacial pain (CFP Group) and 18 patients with only PD (PD Group). Following surgical removal, the tissue underwent routine histological techniques and was stained by immunohistochemistry with antibodies against SP, nNOS and iNOS. Using an image analysis system, we assessed the SP, nNOS and iNOS content in total gingival tissue as well as in both epithelial and connective gingival area. We observed high expression of nNOS in gingival tissue obtained from CFP patients (p<0.001), particularly in the epithelium area (p<0.001) comparatively to PD patients. In addition, the iNOS expression was also increased in the CFP group in the connective gingival tissue (p=0.003). There was no difference concerning SP expression between the groups. Our results suggest that nitric oxide, particularly derived from nNOS, modulates not only PD but also chronic craniofacial pain, since patients with this association presented an increase in nNOS and iNOS expression in gingival tissue.

La enfermedad periodontal (EP) es una patología crónica que pueden tener acción local y sistémica. A pesar de que hay una serie de mediadores inflamatorios implicados en la EP, los mecanismos implicados en el dolor craneofacial crónico asociado con la EP aún no están elucidados. El objetivo fue evaluar la inmunoreactividad de la sustancia P (SP), óxido nítrico sintetasas neuronal (nNOS) e inducible (iNOS) en muestras gingivales de pacientes con enfermedad periodontal severa con y sin dolor craneofacial crónico. Fueron obtenidas muestras gingivales durante la cirugía periodontal rutinaria de 20 pacientes que presentaron con EP y dolor craneofacial crónico (Grupo PPC) y 18 pacientes sólo con PD (Grupo PD). Después de la extirpación quirúrgica, el tejido se sometió a las técnicas histológicas de rutina y se tiñó por inmunohistoquímica con anticuerpos contra el SP, nNOS e iNOS. Se evaluaron el contenido de SP, nNOS e iNOS en el tejido gingival total, así como la superficie gingival, epitelio y tejido conectivo mediante análisis de imagen. Se observó alta expresión de nNOS en el tejido gingival obtenido a partir de pacientes PPC (p<0,001) en comparación a los pacientes con EP, particularmente en el área de epitelio (p<0,001). Además, la expresión de iNOS se incrementó en el tejido conjuntivo gingival (p= 0,003) del grupo PPC. No hubo diferencia en la expresión de SP entre los grupos. Nuestros resultados sugieren que el óxido nítrico, en particular derivado de nNOS, modula no sólo PD, sino también el dolor craneofacial crónico, ya que los pacientes con esta asociación presentan un aumento de la expresión de nNOS e iNOS en el tejido gingival.

Stress amplifies lung tissue mechanics, inflammation and oxidative stress induced by chronic inflammation.

BACKGROUND: Mechanisms linking behavioral stress and inflammation are poorly understood, mainly in distal lung tissue. OBJECTIVE: We have investigated whether the forced swim stress (FS) could modulate lung tissue mechanics, iNOS, cytokines, oxidative stress activation, eosinophilic recruitment, and remodeling in guinea pigs (GP) with chronic pulmonary inflammation. METHODS: The GP were exposed to ovalbumin or saline aerosols (2×/wk/4wks, OVA, and SAL). Twenty-four hours after the 4th inhalation, the GP were submitted to the FS protocol (5×/wk/2wks, SAL-S, and OVA-S). Seventy-two hours after the 7th inhalation, lung strips were cut and tissue resistance (Rt) and elastance (Et) were obtained (at baseline and after OVA and Ach challenge). Strips were submitted to histopathological evaluation. RESULTS: The adrenals' weight, the serum cortisol, and the catecholamines were measured. There was an increase in IL-2, IL-5, IL-13, IFN-γ, iNOS, 8-iso-PGF2α, and in %Rt and %Et after Ach challenge in the SAL-S group compared to the SAL one. The OVA-S group has had an increase in %Rt and %Et after the OVA challenge, in %Et after the Ach and in IL-4, 8-iso-PGF2α, and actin compared to the OVA. Adrenal weight and cortisol serum were increased in stressed animals compared to nonstressed ones, and the catecholamines were unaltered. CONCLUSION & CLINICAL RELEVANCE: Repeated stress has increased distal lung constriction, which was associated with an increase of actin, IL-4, and 8-iso-PGF2α levels. Stress has also induced an activation of iNOS, cytokines, and oxidative stress pathways.

Repeated stress reduces mucociliary clearance in animals with chronic allergic airway inflammation.

We evaluated if repeated stress modulates mucociliary clearance and inflammatory responses in airways of guinea pigs (GP) with chronic inflammation. The GP received seven exposures of ovalbumin or saline 0.9%. After 4th inhalation, animals were submitted to repeated forced swim stressor protocol (5x/week/2 weeks). After 7th inhalation, GP were anesthetized. We measured transepithelial potential difference, ciliary beat frequency, mucociliary transport, contact angle, cough transportability and serum cortisol levels. Lungs and adrenals were removed, weighed and analyzed by morphometry. Ovalbumin-exposed animals submitted to repeated stress had a reduction in mucociliary transport, and an increase on serum cortisol, adrenals weight, mucus wettability and adhesivity, positive acid mucus area and IL-4 positive cells in airway compared to non-stressed ovalbumin-exposed animals (p<0.05). There were no effects on eosinophilic recruitment and IL-13 positive cells. Repeated stress reduces mucociliary clearance due to mucus rheological-property alterations, increasing acid mucus and its wettability and adhesivity. These effects seem to be associated with IL-4 activation.

Inducible nitric oxide synthase inhibition attenuates lung tissue responsiveness and remodeling in a model of chronic pulmonary inflammation in guinea pigs.

We evaluated the influence of iNOS-derived NO on the mechanics, inflammatory, and remodeling process in peripheral lung parenchyma of guinea pigs with chronic pulmonary allergic inflammation. Animals treated or not with 1400 W were submitted to seven exposures of ovalbumin in increasing doses. Seventy-two hours after the 7th inhalation, lung strips were suspended in a Krebs organ bath, and tissue resistance and elastance measured at baseline and after ovalbumin challenge. The strips were submitted to histopathological measurements. The ovalbumin-exposed animals showed increased maximal responses of resistance and elastance (p<0.05), eosinophils counting (p<0.001), iNOS-positive cells (p<0.001), collagen and elastic fiber deposition (p<0.05), actin density (p<0.05) and 8-iso-PGF2alpha expression (p<0.001) in alveolar septa compared to saline-exposed ones. Ovalbumin-exposed animals treated with 1400 W had a significant reduction in lung functional and histopathological findings (p<0.05). We showed that iNOS-specific inhibition attenuates lung parenchyma constriction, inflammation, and remodeling, suggesting NO-participation in the modulation of the oxidative stress pathway.

Oral tolerance attenuates airway inflammation and remodeling in a model of chronic pulmonary allergic inflammation.

We investigated the effects of oral tolerance (OT) in controlling inflammatory response, hyperresponsiveness and airway remodeling in guinea pigs (GP) with chronic allergic inflammation. Animals received seven inhalations of ovalbumin (1-5mg/mL-OVA group) or normal saline (NS group). OT was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st ovalbumin inhalation (OT1 group) or after the 4th (OT2 group). The induction of OT in sensitized animals decreased the elastance of respiratory system (Ers) response after both antigen and methacholine challenges, peribronchial edema formation, eosinophilic airway infiltration, eosinophilopoiesis, and airways collagen and elastic fiber content compared to OVA group (P<0.05). The number of mononuclear cells and resistance of respiratory system (Rrs) responses after antigen and methacholine challenges were decreased only in OT2 group compared to OVA group (P<0.05). Concluding, our results show that inducing OT attenuates airway remodeling as well as eosinophilic inflammation and respiratory system mechanics.

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs.

Recent studies emphasize the presence of alveolar tissue inflammation in asthma. Immunotherapy has been considered a possible therapeutic strategy for asthma, and its effect on lung tissue had not been previously investigated. Measurements of lung tissue resistance and elastance were obtained before and after both ovalbumin and acetylcholine challenges. Using morphometry, we assessed eosinophil and smooth muscle cell density, as well as collagen and elastic fiber content, in lung tissue from guinea pigs with chronic pulmonary allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/ml; OVA group) or saline (SAL group) during 4 wk. Oral tolerance (OT) was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st inhalation (OT1 group) or after the 4th (OT2 group). The ovalbumin-exposed animals presented an increase in baseline and in postchallenge resistance and elastance related to baseline, eosinophil density, and collagen and elastic fiber content in lung tissue compared with controls. Baseline and post-ovalbumin and acetylcholine elastance and resistance, eosinophil density, and collagen and elastic fiber content were attenuated in OT1 and OT2 groups compared with the OVA group. Our results show that inducing oral tolerance attenuates lung tissue mechanics, as well as eosinophilic inflammation and extracellular matrix remodeling induced by chronic inflammation.

Effects of chronic L-NAME treatment lung tissue mechanics, eosinophilic and extracellular matrix responses induced by chronic pulmonary inflammation.

The importance of lung tissue in asthma pathophysiology has been recently recognized. Although nitric oxide mediates smooth muscle tonus control in airways, its effects on lung tissue responsiveness have not been investigated previously. We hypothesized that chronic nitric oxide synthase (NOS) inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME) may modulate lung tissue mechanics and eosinophil and extracellular matrix remodeling in guinea pigs with chronic pulmonary inflammation. Animals were submitted to seven saline or ovalbumin exposures with increasing doses (1 approximately 5 mg/ml for 4 wk) and treated or not with L-NAME in drinking water. After the seventh inhalation (72 h), animals were anesthetized and exsanguinated, and oscillatory mechanics of lung tissue strips were performed in baseline condition and after ovalbumin challenge (0.1%). Using morphometry, we assessed the density of eosinophils, neuronal NOS (nNOS)- and inducible NOS (iNOS)-positive distal lung cells, smooth muscle cells, as well as collagen and elastic fibers in lung tissue. Ovalbumin-exposed animals had an increase in baseline and maximal tissue resistance and elastance, eosinophil density, nNOS- and iNOS-positive cells, the amount of collagen and elastic fibers, and isoprostane-8-PGF(2alpha) expression in the alveolar septa compared with controls (P<0.05). L-NAME treatment in ovalbumin-exposed animals attenuated lung tissue mechanical responses (P<0.01), nNOS- and iNOS-positive cells, elastic fiber content (P<0.001), and isoprostane-8-PGF(2alpha) in the alveolar septa (P<0.001). However, this treatment did not affect the total number of eosinophils and collagen deposition. These data suggest that NO contributes to distal lung parenchyma constriction and to elastic fiber deposition in this model. One possibility may be related to the effects of NO activating the oxidative stress pathway.

Capsaicin-sensitive nerves and neurokinins modulate non-neuronal nNOS expression in lung.

We investigated the effects of substance P (SP) and neurokinin A (NKA) infusion and acute stimulation of capsaicin-sensitive sensory nerves fibers (CAP) on lung recruitment of neuronal nitric oxide synthase (nNOS)-positive inflammatory and respiratory epithelial (RE) cells in guinea-pigs. We evaluated if the effects of CAP stimulation were maintained until 14 days and had functional pulmonary repercussions. After 24h of CAP and 30 min after SP and NKA infusions there was an increase in nNOS-positive eosinophils and mononuclear cells compared to controls (P<0.05). SP group presented an increase in nNOS-positive RE (P<0.05). After 14 days of CAP stimulation, there was a reduction in resistance (R(rs)) and elastance (E(rs)) of respiratory system in capsaicin pre-treated animals. We noticed a correlation between nNOS-positive eosinophils (R=-0.644, P<0.05) and mononuclear cells (R=-0.88, P<0.001) and R(rs). Concluding, CAP and neurokinins increase nNOS expression by inflammatory and RE cells. The increase in nNOS expression induced by low and high doses stimulation of CAP is longstanding and correlated to pulmonary mechanical repercussions.

Effects of stress and neuropeptides on airway responses in ovalbumin-sensitized rats.

OBJECTIVE: The aim of this study was to investigate the influence of stress and neuropeptides on airway responses in ovalbumin (OVA)-sensitized rats. METHODS: Three experimental conditions were employed: neonatal capsaicin treatment, foot shock stress and OVA sensitization. For neuropeptide depletion, male Wistar rats were neonatally treated with capsaicin (50 mg/kg) or with control solution 2 days after birth. Ninety days later, they were injected with OVA and aluminum hydroxide (ED0) or no injection. Thereafter, rats of the stressed groups were individually placed in a shuttle box where they received 50 mild escapable foot shocks/day; the stressful stimuli were repeated until ED14, when the animals received OVA aerosol. Pulmonary mechanic function was measured before and after OVA challenge in anesthetized and mechanically ventilated rats. RESULTS: Data on ultrasonic vocalizations and corticosterone showed high levels of anxiety in stressed animals. As expected, a significant increment in airway elastance and resistance after the OVA challenge was found in sensitized rats compared to non-sensitized ones. Capsaicin treatment decreased the values of elastance in sensitized and non-stressed rats; however, after the OVA challenge, elastance was increased in stressed animals. No differences were found in the levels of resistance among sensitized and non-stressed rats; however, a reduced increment in resistance was verified in capsaicin-treated, stressed animals. CONCLUSIONS: Our results suggest that neurokinin depletion and stress may affect smooth muscle tonus around the airways during an anaphylactic reaction. These data suggest that stress and neuropeptides play a significant role in pulmonary function in OVA-sensitized rats.

Morphological and functional determinants of fluoxetine (Prozac)-induced pulmonary disease in an experimental model.

Fluoxetine treatment effects were determined by evaluating respiratory mechanics (elastance/resistance) and exhaled nitric oxide, as well as mononuclear and polymorphonuclear cell recruitment into the lungs, in an experimental guinea pig model. Guinea pigs were divided into four groups: Fl (fluoxetine only, n=7); Fl+Sw (fluoxetine and forced swimming, n=7); Ns+Sw (normal saline and forced swimming, n=8); and Ns (normal saline only, n=8). Treated animals received oral fluoxetine (10 mg/(kg day)) for 30 consecutive days. On day 31, all animals were anesthetized and mechanically ventilated so that respiratory system elastance and resistance, as well exhaled nitric oxide, could be determined. The lungs were then excised en bloc for histological and immunohistochemical evaluation. Forced swimming induced bronchodilation in untreated animals and bronchoconstriction in fluoxetine-treated animals. Fluoxetine treatment was also associated with mononuclear infiltration (predominantly into alveolar walls) and neutrophil recruitment. In addition, levels of exhaled nitric oxide, an inflammatory marker, were higher in fluoxetine-treated animals. Swimming-induced stress also amplified mononuclear cell recruitment to the lungs. These results show that, in this experimental model, fluoxetine treatment reproduces the pathology of chronic interstitial pneumonia in humans.

Effects of nitric oxide synthases in chronic allergic airway inflammation and remodeling.

The precise role of each nitric oxide (NO) synthase (NOS) isoform in the pathobiology of asthma is not well established. Our objective was to investigate the contribution of constitutive NO synthase (cNOS) and inducible NOS (iNOS) isoforms to lung mechanics and inflammatory and remodeling responses in an experimental model of chronic allergic pulmonary inflammation. Guinea pigs were submitted to seven ovalbumin exposures with increasing doses (1 approximately 5 mg/ml) for 4 wk. The animals received either chronic L-NAME (N-nitro-L-arginine methyl ester, in drinking water) or 1,400 W (iNOS-specific inhibitor, intraperitoneal) treatments. At 72 h after the seventh inhalation of ovalbumin solution, animals were anesthetized, mechanically ventilated, exhaled NO was collected, and lung mechanical responses were evaluated before and after antigen challenge. Both L-NAME and 1,400 W treatments increased baseline resistance and decreased elastance of the respiratory system in nonsensitized animals. After challenge, L-NAME increased resistance of the respiratory system and collagen deposition on airways, and decreased peribronchial edema and mononuclear cell recruitment. Administration of 1,400 W reduced resistance of the respiratory system response, eosinophilic and mononuclear cell recruitment, and collagen and elastic fibers content in airways. L-NAME treatment reduced both iNOS- and neuronal NOS-positive eosinophils, and 1,400 W diminished only the number of eosinophils expressing iNOS. In this experimental model, inhibition of NOS-derived NO by L-NAME treatment amplifies bronchoconstriction and increases collagen deposition. However, blockage of only iNOS attenuates bronchoconstriction and inflammatory and remodeling processes.

Effects of acute and chronic nitric oxide inhibition in an experimental model of chronic pulmonary allergic inflammation in guinea pigs.

Endogenously produced nitric oxide is a recognized regulator of physiological lung events, such as a neurotransmitter and a proinflammatory mediator. We tested the differences between chronic and acute nitric oxide inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME) treatment in lung mechanics, inflammation, and airway remodeling in an experimental asthma model in guinea pigs. Both acute and chronic L-NAME treatment reduced exhaled nitric oxide in sensitized animals (P < 0.001). Chronic L-NAME treatment increased baseline and maximal responses after antigen challenge of respiratory system resistance and reduced peribronchial edema and mononuclear cells airway infiltration (P < 0.05). Acute administration of L-NAME increased maximal values of respiratory system elastance and reduced mononuclear cells and eosinophils in airway wall (P < 0.05). Chronic ovalbumin exposure resulted in airway wall thickening due to an increase in collagen content (P < 0.005). Chronic nitric oxide inhibition increased collagen deposition in airway wall in sensitized animals (P < 0.05). These data support the hypothesis that in this model nitric oxide acts as a bronchodilator, mainly in proximal airways. Furthermore, chronic nitric oxide inhibition was effective in reducing edema and mononuclear cells in airway wall. However, airway eosinophilic inflammation was unaltered by chronic L-NAME treatment. In addition, nitric oxide inhibition upregulates collagen deposition in airway walls.

Neurokinins and inflammatory cell iNOS expression in guinea pigs with chronic allergic airway inflammation.

In the present study we evaluated the role of neurokinins in the modulation of inducible nitric oxide synthase (iNOS) inflammatory cell expression in guinea pigs with chronic allergic airway inflammation. In addition, we studied the acute effects of nitric oxide inhibition on this response. Animals were anesthetized and pretreated with capsaicin (50 mg/kg sc) or vehicle 10 days before receiving aerosolized ovalbumin or normal saline twice weekly for 4 wk. Animals were then anesthetized, mechanically ventilated, given normal saline or N(G)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg ic), and challenged with ovalbumin. Prechallenge exhaled NO increased in ovalbumin-exposed guinea pigs (P < 0.05 compared with controls), and capsaicin reduced this response (P < 0.001). Compared with animals inhaled with normal saline, ovalbumin-exposed animals presented increases in respiratory system resistance and elastance and numbers of total mononuclear cells and eosinophils, including those expressing iNOS (P < 0.001). Capsaicin reduced all these responses (P < 0.05) except for iNOS expression in eosinophils. Treatment with l-NAME increased postantigen challenge elastance and restored both resistance and elastance previously attenuated by capsaicin treatment. Isolated l-NAME administration also reduced total eosinophils and mononuclear cells, as well as those cells expressing iNOS (P < 0.05 compared with ovalbumin alone). Because l-NAME treatment restored lung mechanical alterations previously attenuated by capsaicin, NO and neurokinins may interact in controlling airway tone. In this experimental model, NO and neurokinins modulate eosinophil and lymphocyte infiltration in the airways.

Effects of neurokinins on airway and alveolar eosinophil recruitment.

The authors assessed the role of substance P (SP) and neurokinin A (NKA) and their receptor antagonists (RAs) SR140333 and SR48968 (respectively for NK(1) and NK(2) receptors) in pulmonary eosinophil influx induced by stimulation of capsaicin (CAP)-sensitive nerve terminals. The increase in respiratory system resistance after capsaicin infusion was attenuated by NK(2)RA and association of NK(1)NK(2)RA (P<.001). Respiratory system elastance (Ers) increase was attenuated with use of NK(1)NK(2)RA (P<.001). In alveolar wall, there was an increase in eosinophils after 30 minutes of CAP infusion (P<.001) and was attenuated after 24 hours. Pretreatment with NK(1)RA, NK(2)RA, and NK(1)NK(2)RA decreased eosinophils in alveolar wall (P<.001). SP induced an increase of eosinophils in alveolar wall (P<.001), although NKA may also contribute to this response. In airway wall, the authors observed an increase of eosinophils at 30 minutes (P=.006) till 24 hours after CAP infusion. They noticed a predominant influx of cells around airway wall after CAP and SP infusion. Pretreatment with NK(1)RA and NK(1)NK(2)RA reduced eosinophils (P<.001) in airway wall. Both SP and NKA contribute to eosinophil lung recruitment in distal airways and in alveolar wall, and these findings suggest that neurokinins may contribute to the development of eosinophilic inflammation in both allergic asthma and hypersensitivity pneumonitis.

Effects of diazepam and stress on lung inflammatory response in OVA-sensitized rats.

The influence of stress and diazepam treatment on airway inflammation was investigated in ovalbumin (OVA)-sensitized rats. Animals were injected with OVA plus aluminum hydroxide intraperitoneally (day 0) and boosted with OVA subcutaneously (day 7). From the first to 13th day after sensitization, rats were treated with diazepam, and 1 h later they were placed in a shuttle box where they received 50 mild escapable foot shocks/day preceded by a sound signal (S). Response during the warning (S) canceled shock delivery and terminated the S. On day 14, rats were submitted to a single session of 50 inescapable foot shocks preceded by S and then were challenged with OVA. High levels of stress were detected in shocked animals, manifested as ultrasonic vocalizations. Morphometric analysis of stressed animals revealed a significant increase in both edema and lymphomononucleated cells in airways compared with controls. Diazepam treatment reduced edema in stressed and nonstressed rats. No differences were found in polymorphonucleated cell infiltration. Diazepam treatment reduced lymphomononucleated cell infiltration in stressed animals. These data suggest that stress and diazepam treatment play relevant roles in edema and lymphomononucleated airway inflammation in OVA-sensitized rats.