Spine degeneration in a murine model of chronic human tobacco smokers.

OBJECTIVE: To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS: Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS: Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS: These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

Effects of mannitol on the acute lung injury induced by oleic acid in rats.

Our purpose was to evaluate the pulmonary effects of mannitol infusion in a rat model of acute lung injury induced by oleic acid (OA) to compare the effects of mannitol to those of another diuretic, furosemide (FUR), and to assess if mannitol effects remained after correction of the volume depletion induced by this agent. Acute lung injury was induced in Wistar rats by intravenous administration of 100 mg/kg of OA. Mannitol (1 mL of a 20% solution) was infused either 15 min before or 2 h after OA infusion. FUR was infused intravenously in a dose (1 mg/kg) that induced a similar amount of diuresis compared to mannitol. We also studied rats that received NaCl 0.9% infusion to correct for volume losses induced by mannitol. The severity of the acute lung injury was evaluated by morphometric studies of the lungs 4 h after OA infusion. The amount of intraalveolar fluid accumulation and the intensity of alveolar distention and collapse were evaluated. Mannitol infusion either 15 min before or 2 h after OA administration resulted in a significant decrease in the amount of intraalveolar edema and alveolar distention and collapse (P < 0.001). FUR administration before OA infusion had an effect similar to mannitol. We did not observe any significant effect of mannitol when the rats received saline infusion to correct for diuresis induced by mannitol. We conclude that mannitol decreases the severity of pulmonary injury induced by OA in rats. This effect is mainly due to its diuretic properties.

Exhaled nitric oxide collected with two different mouthpieces: a study in asthmatic patients.

Techniques for collecting exhaled nitric oxide (ENO) recommend the use of antibacterial filters of 0.3 m. The aim of the present study was to compare the measurements of ENO obtained with two different filtering devices. Air samples from 17 asthmatic and 17 non-asthmatic subjects were collected by a recommended off-line technique using two different mouthpieces: 1) the Sievers disposable tool (A) under a breathing pressure of 18 cmH2O, and 2) a mouthpiece containing a HEPA filter (B) under a breathing pressure of 12 cmH2O. The nitric oxide samples were collected into an impermeable reservoir bag. Values for ENO were compared using two-way repeated measures ANOVA followed by the Tukey test. Agreement was assessed by Bland-Altman analysis. ENO values obtained with mouthpieces A and B were comparable for asthmatic (mean +/- SEM, 42.9 +/- 6.9 vs 43.3 +/- 6.6 ppb) and non-asthmatic (13.3 +/- 1.3 vs 13.7 +/- 1.1 ppb) subjects. There was a significant difference in ENO between asthmatics and non-asthmatics using either mouthpiece A (P<0.001) or B (P<0.001). There was a positive correlation between mouthpiece A and mouthpiece B for both groups. The Bland-Altman limits of agreement were considered to be acceptable. Mouthpiece B was less expensive than A, and these data show that it can be used without compromising the result. Our data confirm reports of higher ENO values in the presence of airway inflammation.

Capsaicin-induced release of VIP-like immunoreactivity: modulation by enzyme inhibitors.

Vasoactive intestinal peptide (VIP) is a potent bronchial smooth muscle relaxant. In the present study we measured the release of VIP-like immunoreactivity (VIP-LI) after tracheal infusion of capsaicin, histamine, and methacholine in isolated guinea pig lungs superfused through the trachea. We also studied if inhibition of VIP enzymatic cleavage using a combination of an inhibitor of neutral endopeptidase [thiorphan (Thio)] and an inhibitor of serine proteases [soybean trypsin inhibitor (STI)] influenced the airway effects of capsaicin. Infusion of capsaicin resulted in a significant increase in VIP-LI in the perfusate (12.32 +/- 4.80 to 33.52 +/- 8.46 fmol/5 min fraction; P < 0.001). There was no increase in VIP-LI after infusion of methacholine or histamine. Maximal changes in airway opening pressure (Pao) observed 0-10 min after tracheal infusion of capsaicin were significantly greater in the Thio group than the control group and the groups of lungs that received STI or STI + Thio (P < 0.005). In addition, recovery of VIP-LI in the superfusate after infusion of capsaicin was significantly greater in the group of lungs that was superfused with Thio + STI compared with STI, Thio, and control groups. Our results suggest that a bronchodilator peptide with the profile of enzymatic cleavage of VIP also modulates capsaicin effects, since the increase in Pao in the presence of Thio + STI was significantly lower than Thio alone.

Acute lung injury in two experimental models of acute pancreatitis: infusion of saline or sodium taurocholate into the pancreatic duct.

OBJECTIVE: To compare acute pulmonary changes secondary to sodium taurocholate hemorrhagic pancreatitis with those changes secondary to a less severe pancreatitis induced by saline infusion into the biliopancreatic duct. DESIGN: Prospective, randomized controlled trial. SETTING: University pulmonary laboratory. SUBJECTS: A total of 110 male Wistar rats. INTERVENTIONS: Pancreatitis was induced by either 0.5 mL of a 4% solution of sodium taurocholate (TAU group) or 0.5 mL of normal saline (SAL group) injection into the biliopancreatic duct. Data were compared with data from control (sham-operated) animals (SHAM group). MEASUREMENTS AND MAIN RESULTS: The severity of pancreatic and pulmonary injuries was evaluated 1, 3, and 8 days after the induction of acute pancreatitis by morphometric and pulmonary mechanical studies. Biliopancreatic duct pressure was measured during infusion of solutions in SAL and TAU groups. SAL and TAU groups developed an intense necrohemorrhagic pancreatitis on day 1 without differences in biliopancreatic duct pressures (134.0+/-45.1 cm H2O vs. 123.3+/-23.4 cm H2O). Acute pancreatic lesions were still intense on day 3 in the TAU group only. Pulmonary resistance in SAL and TAU groups was significantly greater than in the SHAM group on day 3 only. On day 1, there was an increase in intraalveolar edema in both groups (p < .02). There was an increase in polymorphonuclear cells in alveolar septa on day 1 only in the TAU group (p < .001). In contrast, both experimental groups presented greater values of PMN cells on day 8 compared with the SHAM group (p < .001). Both groups with pancreatitis showed an increase in alveolar distention and collapse on day 1 that persisted only in the TAU group on days 3 and 8. No deaths were observed in the control (SHAM) group. In contrast, the SAL group had lower mortality than the TAU group in the first two days (17% and 52%, respectively, p = .03). CONCLUSION: High-pressure infusion of normal saline into the biliopancreatic duct of rats results in significant pancreatic and lung alterations. These changes are worse in the presence of sodium taurocholate.

Airway responses to capsaicin in guinea pigs: role of NK-1 and NK-2 neurokinin receptors.

The role of NK-1 and NK-2 receptors on the pulmonary response to capsaicin in guinea pigs was evaluated using intravenous infusion of selective nonpeptide antagonists of NK 1 (CP 96345, 300 nmol/kg, and SR 140333, 300 nmol/kg) and NK-2 (SR 48968, 100 nmol/kg) neurokinin receptors. Maximal values of pulmonary dynamic elastance (Edyn) and pulmonary resistance (RL) after capsaicin infusion were significantly lower in the presence of SR 48968 (p < .005). Morphometric analysis of lungs obtained by quick-freezing showed significant attenuation of airway contraction and peribronchiolar edema formation in the presence of NK-2 antagonist (p < .001). When compared to guinea pigs that received only capsaicin, animals that received SR 140333 or CP 96345 showed lower values of Edyn, RL, airway contraction, and peribronchiolar edema, but only the difference in Edyn values was significant. The combination of NK-1 and NK-2 antagonists was not more effective than NK-2 antagonist alone in attenuating capsaicin effects. The results suggest that airway effects of capsaicin are mainly mediated by activation of NK-2 receptors although NK-1 receptors may also play a role.

Neurokinin depletion attenuates pulmonary changes induced by antigen challenge in sensitized guinea pigs.

The role of neurokinins in the acute pulmonary response to antigen was studied in guinea pigs that received ovalbumin (50 mg/kg ip) on days 1 and 3 and capsaicin (50 mg/kg sc) on day 21 (OAC); ovalbumin on days 1 and 3 (OA1); capsaicin on day 1 and OA on days 8 and 10 (COA); and ovalbumin on days 8 and 10 (OA2). On day 28, guinea pigs were submitted to ovalbumin aerosol challenge. Maximal values of pulmonary dynamic elastance (Edyn) and pulmonary resistance (RL) were significantly lower in OAC and COA groups compared with OA1 and OA2 groups (P < 0.001). There was no difference between maximal Edyn and RL values obtained in OAC and COA groups. Morphometric analysis of lungs showed significantly (P < 0.05) lower values of contraction index of airways, peribronchial edema, and alveoli over inflation in guinea pigs that received capsaicin compared with intact guinea pigs. Capsaicin treatment did not influence the formation of specific IgG1 anaphylactic antibodies. We conclude that neurokinin depletion results in a decrease in the pulmonary mechanical and inflammatory responses to antigen challenge in sensitized guinea pigs. These effects are observed when capsaicin is given either before or after sensitization.

Neonatal capsaicin treatment decreases airway and pulmonary tissue responsiveness to methacholine.

We studied the effects of selective depletion of neurokinins in sensory nerve fibers by capsaicin treatment on the airway and pulmonary tissue responses to methacholine. Dose-response curves to aerosolized methacholine were performed on anesthetized and mechanically ventilated Wistar rats. Capsaicin (50 mg/kg sc) was administered to 2-day-old rats, and the animals were studied after 12 wk. The response to each dose of methacholine was determined by measuring changes in airway resistance (R(aw)), dynamic pulmonary elastance (Edyn), and pulmonary tissue resistance (Rtis). We calculated sensitivity (Kx) as the concentration of methacholine required for a one-half maximal response and reactivity as the relationship between the maximum response and Kx. Capsaicin treatment resulted in significantly greater values of Kx and lower values of reactivity for R(aw), Edyn, and Rtis compared with control rats. Morphometric analysis of airways showed similar values of the area occupied by smooth muscle but a significantly lower (P < 0.02) area of airway epithelium in capsaicin-treated rats. Our results suggest that methacholine requires capsaicin-sensitive nerves for part of its airway and lung tissue effects.

C-ANCA-positive IgG fraction from patients with Wegener's granulomatosis induces lung vasculitis in rats.

The aim of the present study was to analyse in rats the ability of C-ANCA-positive IgG fraction in triggering inflammatory response on pulmonary tissue. Wistar rats (n = 18) were injected via the the internal jugular vein with 20 mg of total C-ANCA-positive IgG fraction isolated from serum of three different Wegener's granulomatosis patients obtained before therapy. Similarly, control rats were treated with IgG fraction from two rheumatoid arthritis patients (n = 7), IgG from six normal human sera (n = 15) or saline (n = 18), respectively. Animals were sacrificed after 24h of injection for histological analysis of the lungs. Vasculitis and inflammatory infiltrate were consistently absent in rats injected with rheumatoid arthritis IgG or saline and in 14/15 of normal IgG treated animals. In contrast, marked vasculitis was observed in all 18 animals injected with C-ANCA-positive IgG fraction. The histological features were characterized by the presence of a perivascular pleomorphic cellular sheath, particularly around small vessels, endothelial adherence and diapedesis of polymorphonuclear leucocytes and presence of granuloma-like lesions. A dose-response relationship was observed between protein concentration of C-ANCA IgG sample and the intensity of the inflammatory response in the animals. In addition, IgG fraction with undetectable C-ANCA, obtained from one patient in remission after treatment, was not able to reproduce the pulmonary tissue alterations induced by its paired IgG that was positive for C-ANCA taken before therapy. The experimental model described herein may be useful to characterize more effectively the pathogenic mechanism of C-ANCA in Wegener's disease.

Exhaled nitric oxide collected with two different mouthpieces: a study in asthmatic patients

Techniques for collecting exhaled nitric oxide (ENO) recommend the use of antibacterial filters of 0.3 æm. The aim of the present study was to compare the measurements of ENO obtained with two different filtering devices. Air samples from 17 asthmatic and 17 non-asthmatic subjects were collected by a recommended off-line technique using two different mouthpieces: 1) the Sievers disposable tool (A) under a breathing pressure of 18 cmH2O, and 2) a mouthpiece containing a HEPA filter (B) under a breathing pressure of 12 cmH2O. The nitric oxide samples were collected into an impermeable reservoir bag. Values for ENO were compared using two-way repeated measures ANOVA followed by the Tukey test. Agreement was assessed by Bland-Altman analysis. ENO values obtained with mouthpieces A and B were comparable for asthmatic (mean ± SEM, 42.9 ± 6.9 vs 43.3 ± 6.6 ppb) and non-asthmatic (13.3 ± 1.3 vs 13.7 ± 1.1 ppb) subjects. There was a significant difference in ENO between asthmatics and non-asthmatics using either mouthpiece A (P<0.001) or B (P<0.001). There was a positive correlation between mouthpiece A and mouthpiece B for both groups. The Bland-Altman limits of agreement were considered to be acceptable. Mouthpiece B was less expensive than A, and these data show that it can be used without compromising the result. Our data confirm reports of higher ENO values in the presence of airway inflammation