Naomi Kondo Nakagawa

Experimental Models Of Sepsis And Their Clinical Relevance

Sepsis remains a major cause of morbidity and mortality mainly because of sepsis-induced multiple organ dysfunction. In contrast to preclinical studies, most clinical trials of promising new treatment strategies for sepsis have failed to demonstrate efficacy. Although many reasons could account for this discrepancy, the misinterpretation of preclinical data obtained from experimental studies and especially the use of animal models that do not adequately mimic human sepsis may have been contributing factors. In this review, the potentials and limitations of various animal models of sepsis are discussed to clarify to which extent these findings are relevant to human sepsis. Such models include intravascular infusion of endotoxin or live bacteria, bacterial peritonitis, cecal ligation and perforation, soft tissue infection, pneumonia or meningitis models using different animal species including rats, mice, rabbits, dogs, pigs, sheep, and nonhuman primates. Despite several limitations, animal models remain essential in the development of all new therapies for sepsis and septic shock because they provide fundamental information about the pharmacokinetics, toxicity, and mechanism of drug action that cannot be replaced by other methods. New therapeutic agents should be studied in infection models, even after the initiation of the septic process. Furthermore, debility conditions need to be reproduced to avoid the exclusive use of healthy animals, which often do not represent the human septic patient.

Effects of a heat and moisture exchanger and a heated humidifier on respiratory mucus in patients undergoing mechanical ventilation.

Objective: To evaluate the effects of a heat and moisture exchanger and a heated humidifier on respiratory mucus and transportability by cilia and cough in patients undergoing invasive mechanical ventilation (up to 72 hrs). Design: Prospective, randomized, clinical study. Setting: General intensive care unit and university research laboratory. Patients: A total of 32 consecutive patients with acute respiratory failure, who were intubated and mechanically ventilated in the intensive care unit setting, were enrolled in the study. Interventions: Patients were randomly assigned to receive as a humidifying system a heat and moisture exchanger (HME) or heated humidified water (HHW) at the onset of mechanical ventilation (time 0). Respiratory mucus samples were collected by suction using a sterile technique at time 0, 24, 48, and 72 hrs of mechanical ventilation. Measurements and Main Results: Eleven patients were excluded from this study because of either extubation or death before 72 hrs of mechanical ventilation, leaving 12 patients in the HME group and nine patients in the HHW group. Ventilatory variables including minute volume, mean airway pressure, positive end‐expiratory pressure, FIO2, as well as PaO2/FIO2 ratio, fluid balance (last 6 hrs), furosemide, and inotrope administration (last 4 hrs) were recorded. In vitro mucus transportability by cilia was evaluated on the mucus‐depleted frog palate model, and the results were expressed as the mucus transport rate. Cough clearance (an estimation of the interaction between the flow of air and the mucus lining the bronchial walls) was measured using a simulated cough machine, the results being expressed in millimeters. Mucus wettability was measured by the contact angle between a mucus sample drop and a flat glass surface. Mucus rheologic properties (mechanical impedance [log G*] and the ratio between viscosity and elasticity [tan δ]) were measured using a magnetic microrheometer at 1 and 100 cGy/sec deformation frequency. The two humidification groups were comparable in terms of the Acute Physiology and Chronic Health Evaluation II score, age, gender, ventilatory variables, fluid balance, use of inotropes, and furosemide. Conclusion: Ours results indicate that air humidification with either HME or HHW at 32°C (89.6°F) has similar effects on mucus rheologic properties, contact angle, and transportability by cilia in patients undergoing mechanical ventilation, except for transportability by cough, which diminished after 72 hrs of mechanical ventilation in the HME group (p = .0441).

LEUKOCYTE-ENDOTHELIUM INTERACTIONS AFTER HEMORRHAGIC SHOCK/REPERFUSION AND CECAL LIGATION/PUNCTURE: AN INTRAVITAL MICROSCOPIC STUDY IN RAT MESENTERY

ABSTRACT Hemorrhagic shock/reperfusion (HS/R) followed by sepsis triggers systemic microcirculatory disturbances that may induce multiple organ failure. The present study evaluated the effects of HS/R and cecal ligation and puncture, followed by necrotic cecal resection/peritoneal lavage (REL) on leukocyte-endothelium interactions at the mesentery. Eighty-one anesthetized Wistar rats (200-250 g) were randomly assigned to a first injury: (1) control-HS-no hemorrhagic shock/no reperfusion group, (2) HS/blood-HS/R with 25% shed blood, and (3) HS/blood + LR-HS/R with 25% of the shed blood + lactated Ringers solution, 3× shed blood volume. Twenty-four hours post-HS/R, animals were submitted to cecal ligation and puncture and, 24 h thereafter, to REL. Leukocyte-endothelium interactions were assessed by intravital microscopy and intercellular adhesion molecule (ICAM) 1 and P-selectin expression by immunohistochemistry. Lungs were observed for ICAM-1 expression and neutrophil infiltration. Single and double injury induced significant increases in rolling (~2-fold), adherent (~5-fold), and migrated leukocytes (~7-fold); ICAM-1 expression (~1/2-fold), and P-selectin expression (~1/2-fold) at the mesentery compared with control-HS group. REL normalized leukocyte-endothelium interactions at the mesentery in single-injured animals. However, in double-injured rats, adherence and migration of leukocytes decreased but did not normalize. Similar results were observed on ICAM-1 expression and neutrophil infiltration in the lungs from these animals. In conclusion, the current in vivo observation of the mesenteric microcirculation after a double injury followed by REL is a suitable model for the systematic evaluation of the inflammatory reaction at local and distant sites. In addition, data presented herein emphasized the importance of surgical removal of the septic focus in controlling the otherwise lethal sepsis-induced multiple organ dysfunction syndrome.

Aging, diabetes, and hypertension are associated with decreased nasal mucociliary clearance.

BACKGROUND We showed previously that nasal mucociliary clearance was decreased in critically ill elderly subjects, most of whom had diabetes mellitus (DM) and/or hypertension (HTN). To determine if these changes were due to the effects of aging, disease, or critical illness, we studied nasal mucociliary clearance and mucus properties in an ambulatory population consisting of young, elderly, and healthy subjects and those with DM, HTN, or both. METHODS Of 440 subjects contacted, 252 entered the study. The subjects were divided into the following groups: (1) healthy (n 5 79, 18-94 years, 50 men) and (2) DM and/or HTN, of which 37 had DM (14-90 years, 12 men), 52 had HTN (23-90 years, 12 men), and 84 had both DM and HTN (25-82 years, 33 men). Subjects were also grouped by age: , 40 years, 40 to 59 years, and 60 years. We assessed demographic and clinical data, quality of life using the 36-Item Short Form Health Survey (SF-36) questionnaire, nasal mucociliary clearance using the saccharine transit test (STT), and in vitro mucus properties by examining the sneeze (high airflow) clearability and contact angle. A logistic regression analysis for prolonged STT . 12 min was used, and we controlled for age, sex, and diseases. RESULTS Subjects aged . 60 years reported a decreased SF-36 physical component relative to other age groups. Sex, BMI, BP, heart rate, pulse oximetry, blood glucose level, and mucus properties were not associated with prolonged STT. Aging and DM and/or HTN independently increased the risk of prolonged STT. CONCLUSIONS Aging and DM, HTN, or both diseases are independently associated with decreased nasal mucociliary clearance. This may predispose toward respiratory infections.

Young “Healthy” Smokers Have Functional and Inflammatory Changes in the Nasal and the Lower Airways

BACKGROUND Smoking is responsible for most COPD. Although people with COPD often have concomitant nasal disease, there are few studies that report physiologic or inflammatory changes in the upper airways in young asymptomatic smokers. We investigated physiologic and inflammatory changes in the nasal and lower airways of young smokers and if these changes were related to smoking history. METHODS Seventy-two subjects aged between 18 and 35 years (32 healthy nonsmokers and 40 young smokers) participated in this study. We measured nasal mucociliary clearance (MCC), nasal mucus surface contact angle, cell counts, myeloperoxidase and cytokine concentrations in nasal lavage fluid, exhaled breath condensate (EBC) pH, and lung function. RESULTS Smokers had faster MCC, an increased number of cells (macrophages, ciliated cells, and goblet cells), increased lavage myeloperoxidase concentration, and decreased EBC pH compared with nonsmokers. There was a significant inverse relationship between pack-year smoking history and EBC pH. There were no differences in lung function or mucus surface properties comparing smokers to nonsmokers. CONCLUSIONS Young adult smokers have functional and inflammatory changes in the nasal and lower airways and these correlate with smoking history. However, in these young smokers, smoking history was not associated with pulmonary function decline, probably because it is unlikely that spirometry detects early physiologic changes in the airways. TRIAL REGISTRY ClinicalTrials.gov; No.: NCT01877291; URL: www.clinicaltrials.gov.

Effects of biomass burning on nasal mucociliary clearance and mucus properties after sugarcane harvesting

OBJECTIVE Biofuel from sugarcane is widely produced in developing countries and is a clean and renewable alternative source of energy. However, sugarcane harvesting is mostly performed after biomass burning. The aim of this study was to evaluate the effects of harvesting after biomass burning on nasal mucociliary clearance and the nasal mucus properties of farm workers. METHODS Twenty seven sugarcane workers (21-45 years old) were evaluated at the end of two successive time-periods: first at the end of a 6-month harvesting period (harvesting), and then at the end of a 3-month period without harvesting (non-harvesting). Nasal mucociliary clearance was evaluated by the saccharine transit test, and mucus properties were analyzed using in vitro mucus contact angle and mucus transportability by sneeze. Arterial blood pressure, heart rate, respiratory rate, pulse oximetry, body temperature, associated illness, and exhaled carbon monoxide were registered. RESULTS Data are presented as mean values (95% confidence interval). The multivariate model analysis adjusted for age, body-mass index, smoking status and years of working with this agricultural practice showed that harvesting yielded prolonged saccharine transit test in 7.83 min (1.88-13.78), increased mucus contact angle in 8.68 degrees (3.18-14.17) and decreased transportability by sneeze in 32.12 mm (-44.83 to -19.42) compared with the non-harvesting period. No significant differences were detected in any of the clinical parameter at either time-period. CONCLUSION Sugarcane harvesting after biomass burning negatively affects the first barrier of the respiratory system in farm workers by impairing nasal mucociliary clearance and inducing abnormal mucus properties.

The time course of vasoconstriction and endothelin receptor A expression in pulmonary arterioles of mice continuously exposed to ambient urban levels of air pollution

The present study aimed to verify the time course of the effects of environmental levels of urban air pollution toxicity on lung arterioles. BALB/c mice (n=56) were continuously exposed to selective chambers equipped with (filtered, F) or without (non-filtered, NF) filter devices for particles and toxic gases for 24h/day, over 14, 21, 30 or 45 days. After exposure, we evaluated the lumen-wall relationship (an estimator of arteriolar narrowing), endothelial nitric oxide synthase (eNOS) and endothelin type A receptor (ETAr) expression in the vascular wall and inflammatory influx of the peribronchiolar area. Concentrations of fine particulate matter (PM<or=2.5 microg/m(3)), nitrogen dioxide (NO(2)), black smoke (BS), humidity and temperature in both the environment and inside the chambers were measured daily. Filters cleared 100% of BS and 97% of PM inside the F chamber. The arteriole wall of the lungs of mice from NF chamber had an increased ETAr expression (p<or=0.042) concomitant to a decrease in the lumen/wall ratio (p=0.02) on the early days of exposure, compared to controls. They also presented a progressive increment of inflammatory influx in the peribronchiolar area during the study (p=0.04) and decrement of the eNOS expression on the 45th day of exposure in both vascular layers (p<or=0.03). We found that after 14 days of exposure, the ambient levels of air pollutants in Sao Paulo induced vasoconstriction that was associated with an increase in ETAr expression. These vascular results do not appear to be coupled to the progressive inflammatory influx in lung tissue, suggesting a down-regulation of vasoconstrictive mechanisms through an imbalance in the cytokines network. It is likely that these responses are protective measures that decrease tissue damage brought about by continuous exposure to air pollutants.

In vivo observation of mesenteric leukocyte-endothelial interactions after cecal ligation/puncture and surgical sepsis source control

PURPOSE Cecal ligation and puncture (CLP) has been used as a useful model for the induction of polymicrobial sepsis. Necrotic tissue resection and peritoneal lavage (REL) are the surgical procedures for controlling perforated appendicitis. The aim of this study was to evaluate leukocyte-endothelial interactions in the rat mesentery in vivo after CLP and REL. METHODS Thirty-seven male Wistar rats (250-300 g) underwent laparotomy and were randomly assigned to the following groups: 1) SHAM; 2) CLP: animals submitted to CLP, 3) CLP+REL: animals submitted to CLP and REL. Mesenteric leukocyte-endothelial interactions were studied by intravital microscopy assessed once in each animal (3-5 postcapillary venules, 15-25 microm diameter) 24 hours after intervention. Follow-up was performed in all animals; this included analysis of glycemia, lactate, hematocrit, white blood cell count as well as a functional score that was the sum of scoring on the following parameters: alertness, mobility, piloerection, diarrhea, encrusted eyes, and dirty nose and tail. RESULTS None of the animals showed significant changes in body weight (265 +/- 20 g) or in hematocrit levels (46% +/- 2%) during the experimental protocol. Compared to SHAM animals, CLP animals showed an increased number of rolling (2x), adherent, and migrating leukocytes (7x) in the mesenteric microcirculation, an increase in blood glucose (136 +/- 8 mg/dL), lactate (3.58 +/- 0.94 mmol/L), white cell count (23,570 +/- 4,991 cells/mm(3)) and functional alterations (score 11 +/- 1), characterized by impaired alertness and mobility, and presence of piloerection, diarrhea, encrusted eyes, and dirty nose and tail. The REL procedure normalized the number of rolling, adherent, and migrated leukocytes in the mesentery; glycemia; lactate; and white blood cell count. The REL procedure also improved the functional score (7 +/- 1). CONCLUSION Local and systemic inflammation was induced by CLP, while REL completely overcame the inflammatory process.

CPAP Has No Effect on Clearance, Sputum Properties, or Expectorated Volume in Cystic Fibrosis

BACKGROUND: Positive expiratory pressure (PEP) is used for airway clearance in cystic fibrosis (CF) patients. Hypertonic saline (HTS) aerosol increases sputum expectoration volume and may improve respiratory secretion properties. CPAP may also be used to maintain airway patency and mobilize secretions. To evaluate if CPAP would increase the beneficial clearance effect of HTS in subjects with CF, we investigated the effects of CPAP alone and CPAP followed by HTS on sputum physical properties and expectoration volume in CF subjects. METHODS: In this crossover study, 15 CF subjects (mean age 19 y old) were randomized to interventions, 48 hours apart: directed coughs (control), CPAP at 10 cm H2O, HTS 7%, and both CPAP and HTS (CPAP+HTS). Sputum collection was performed at baseline and after interventions. Expectorated volume was determined and in vitro sputum properties were analyzed for contact angle and cough clearability. RESULTS: There were no significant differences between any treatment in arterial blood pressure, heart rate, or pulse oximetry, between the 2 time points. HTS and CPAP+HTS improved cough clearability by 50% (P = .001) and expectorated volume secretion by 530% (P = .001). However, there were no differences between control and CPAP on sputum contact angle, cough clearability, or volume of expectorated secretion. CONCLUSIONS: CPAP alone had no effect on mucus clearance, sputum properties, or expectorated volume, and did not potentiate the effect of HTS alone in CF subjects.

Microcirculatory effects of local and remote ischemic preconditioning in supraceliac aortic clamping.

INTRODUCTION Supraceliac aortic clamping in major vascular procedures promotes splanchnic ischemia and reperfusion (I/R) injury that may induce endothelial dysfunction, widespread inflammation, multiorgan dysfunction, and death. We tested the hypothesis that local or remote ischemic preconditioning (IPC) may be protective against injury after supraceliac aortic clamping through the modulation of mesenteric leukocyte-endothelial interactions, as evaluated with intravital microscopy and expression of adhesion molecules. METHODS Fifty-six male Wistar rats (weight, 190 to 250 g), were divided into four groups of 14 rats each: control-sham surgery without aortic occlusion; I/R through supraceliac aortic occlusion for 20 minutes, followed by 120 minutes of reperfusion; local IPC through supraceliac aortic occlusion for two cycles of 5 minutes of ischemia and 5 minutes of reperfusion, followed by the same protocol of the IR group; remote IPC through infrarenal aortic occlusion for two cycles of 10 minutes of ischemia and 10 minutes of reperfusion, followed by the same protocol of the IR group. Seven animals per group were used to evaluate in vivo leukocyte-endothelial interactions in postcapillary venules with intravital microscopy and another seven animals per group were used to collect mesentery samples for immunohistochemistry demonstration of adhesion molecules expression. RESULTS Supraceliac aortic occlusion increased the number of rolling leukocytes with slower velocities and increased the number of adherent leukocytes to the venular surface and leukocyte migration to the interstitium. The expression of P-selectin, E-selectin, and intercellular adhesion molecule-1 was also increased significantly after I/R. Local or remote IPC reduced the leukocyte recruitment in vivo and normalized the expression of adhesion molecules. CONCLUSIONS Local or remote IPC reduces endothelial dysfunction on mesenteric microcirculation caused by I/R injury after supraceliac aortic clamping.