Viviane Ramos Cagido

Lipopolysaccharide-induced lung injury: Role of P2X7 receptor

RATIONALE P2X7 receptors have been involved in inflammatory and immunological responses, and their activation modulates pro-inflammatory cytokines production by LPS-challenged macrophages. OBJECTIVES To determine the role of P2X7R in LPS-induced acute lung injury in mice. METHODS Wild-type (C57BL/6) and P2X7 knockout mice received intratracheal injection of saline or Escherichia coli LPS (60 μg). After 24h, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage was performed, and lungs were harvested for measurement of morphometry, fibers content, inflammatory cells and cytokine expression by histochemistry and immunohistochemistry. RESULTS Compared with saline, LPS increased lung mechanical parameters, mast cell, collagen and fibronectin deposition in lung parenchyma, as well as nitric oxide and lactate dehydrogenase release into bronchoalveolar fluid in wild-type, but not in P2X7R knockout mice. Alveolar collapse, lung influx of polymorphonuclear and CD14(+) cells, as well as TGF-β, MMP-2, and IL-1β release were higher in wild-type than knockout LPS-challenged mice, while MMP-9 release where similar between the two genotypes. LPS increased macrophage immunoreactivity in lung tissue in both genotypes, but macrophages were not activated in the P2X7R knockout mice. Furthermore, LPS administration increased P2X7R immunoexpression in lung parenchyma in wild-type mice, and TLR4 in both wild-type and P2X7R knockout mice. CONCLUSION P2X7 receptors are implicated in the pathophysiology of LPS-induced lung injury, modulating lung inflammatory and functional changes.

Early short-term versus prolonged low-dose methylprednisolone therapy in acute lung injury

The present study compared the effects of early short-term with prolonged low-dose corticosteroid therapy in acute lung injury (ALI). In total, 120 BALB/c mice were randomly divided into five groups. In the control group, saline was intratracheally (i.t.) instilled. In the ALI group, mice received Escherichia coli lipopolysaccharide (10 μg i.t.). ALI animals were further randomised into four subgroups to receive saline (0.1 mL i.v.) or methylprednisolone (2 mg·kg−1 i.v.) at 6 h, 24 h or daily (for 7 days, beginning at day 1). At 1, 3 and 8 weeks, in vivo and in vitro lung mechanics and histology (light and electron microscopy), collagen and elastic fibre content, cytokines in bronchoalveolar lavage fluid and the expression of matrix metalloproteinase (MMP)-9 and -2 were measured. In vivo (static elastance and viscoelastic pressure) and in vitro (tissue elastance and resistance) lung mechanics, alveolar collapse, cell infiltration, collagen and elastic fibre content and the expression of MMP-9 and MMP-2 were increased in ALI at 1 week. Methylprednisolone led to a complete resolution of lung mechanics, avoided fibroelastogenesis and the increase in the expression of MMP-9 and MMP-2 independent of steroid treatment design. Thus, early short-term, low-dose methylprednisolone is as effective as prolonged therapy in acute lung injury.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 microl, ip) and group Ci received a sublethal dose of microcystins (48.2 microg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262%) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161%). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.

Recruitment maneuver: RAMP versus CPAP pressure profile in a model of acute lung injury

We examined whether recruitment maneuvers (RMs) with gradual increase in airway pressure (RAMP) provide better outcome than continuous positive airway pressure (CPAP) in paraquat-induced acute lung injury (ALI). Wistar rats received saline intraperitoneally (0.5 mL, CTRL) or paraquat (15 mg/kg, ALI). Twenty-four hours later lung mechanics [static elastance, viscoelastic component of elastance, resistive, viscoelastic and total pressures] were determined before and after recruitment with 40cmH2O CPAP for 40s or 40-s-long slow increase in pressure up to 40cmH2O (RAMP) followed by 0 or 5 cmH2O PEEP. Fractional area of alveolar collapse and PCIII mRNA were determined. All mechanical parameters and the fraction area of alveolar collapse were higher in ALI compared to CTRL. Only RAMP-PEEP maneuver significantly improved lung mechanics and decreased PCIII mRNA expression (53%) compared with ALI, while both RMs followed by PEEP decreased alveolar collapse. In conclusion, in the present experimental ALI model, RAMP followed by 5cm H2O PEEP yields a better outcome.

Impact of lung remodelling on respiratory mechanics in a model of severe allergic inflammation

We developed a model of severe allergic inflammation and investigated the impact of airway and lung parenchyma remodelling on in vivo and in vitro respiratory mechanics. BALB/c mice were sensitized and challenged with ovalbumin in severe allergic inflammation (SA) group. The control group (C) received saline using the same protocol. Light and electron microscopy showed eosinophil and neutrophil infiltration and fibrosis in airway and lung parenchyma, mucus gland hyperplasia, and airway smooth muscle hypertrophy and hyperplasia in SA group. These morphological changes led to in vivo (resistive and viscoelastic pressures, and static elastance) and in vitro (tissue elastance and resistance) lung mechanical alterations. Airway responsiveness to methacholine was markedly enhanced in SA as compared with C group. Additionally, IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid were higher in SA group. In conclusion, this model of severe allergic lung inflammation enabled us to directly assess the role of airway and lung parenchyma inflammation and remodelling on respiratory mechanics.

Volume-independent elastance: a useful parameter for open-lung positive end-expiratory pressure adjustment.

BACKGROUND:A decremental positive end-expiratory pressure (PEEP) trial after full lung recruitment allows for the adjustment of the lowest PEEP that prevents end-expiratory collapse (open-lung PEEP). For a tidal volume (Vt) approaching zero, the PEEP of minimum respiratory system elastance (PEEPminErs) is theoretically equal to the pressure at the mathematical inflection point (MIP) of the pressure-volume curve, and seems to correspond to the open-lung PEEP in a decremental PEEP trial. Nevertheless, the PEEPminErs is dependent on Vt and decreases as Vt increases. To circumvent this dependency, we proposed the use of a second-order model in which the volume-independent elastance (E1) is used to set open-lung PEEP. METHODS:Pressure-volume curves and a recruitment maneuver followed by decremental PEEP trials, with a Vt of 6 and 12 mL/kg, were performed in 24 Wistar rats with acute lung injury induced by intraperitoneally injected (n = 8) or intratracheally instilled (n = 8) Escherichia coli lipopolysaccharide. In 8 control animals, the anterior chest wall was surgically removed after PEEP trials, and the protocol was repeated. Airway pressure (Paw) and flow (F) were continuously acquired and fitted by the linear single-compartment model (Paw = Rrs·F + Ers·V + PEEP, where Rrs is the resistance of the respiratory system, and V is volume) and the volume-dependent elastance model (Paw = Rrs·F + E1 + E2·V·V + PEEP, where E2·V is the volume-dependent elastance). From each model, PEEPs of minimum Ers and E1 (PEEPminE1) were identified and compared with each respective MIP. The accuracy of PEEPminE1 and PEEPminErs in estimating MIP was assessed by bias and precision plots. Comparisons among groups were performed with the unpaired t test whereas a paired t test was used between the control group before and after chest wall removal and within groups at different Vts. All P values were then corrected for multiple comparisons by the Bonferroni procedure. RESULTS:In all experimental groups, PEEPminErs, but not PEEPminE1, tended to decrease as Vt increased. The difference between MIP and PEEPminE1 exhibited a lower bias compared with the difference between MIP and PEEPminErs (P < 0.001). The PEEPminE1 was always significantly higher than the PEEPminErs (7.7 vs 3.8, P < 0.001) and better approached MIP (7.7 vs 7.3 cm H2O with P = 0.04 at low Vt, and 7.8 vs 7.1 cm H2O with P < 0.001 at high Vt). CONCLUSIONS:PEEPminE1 better identifies the open-lung PEEP independently of the adjusted Vt, and may be a practical, more individualized approach for PEEP titration.

Effects of dexmedetomidine on respiratory mechanics and control of breathing in normal rats

Dexmedetomidine is a highly selective and specific alpha(2)-adrenergic agonist, with sedative, analgesic, and sympatholytic activities. The aim of the present study was to define the effects of DMED in respiratory mechanics in normal rats. In addition, lung morphometry was studied to determine whether the physiological changes reflected underlying morphological changes defining the sites of action of dexmedetomidine. Arterial blood gases were also determined. Twelve adult Wistar rats were randomly assigned to two groups of six animals each: PENTO and DMED. In PENTO group animals were sedated (diazepam, 5mg, i.p.) and anaesthetised with pentobarbital sodium (20mgkg(-1) i.p.). The rats of the DMED group received dexmedetomidine (250mugkg(-1) i.p. followed by intravenous infusion of 0.5mugkg(-1)h(-1)). In spontaneously breathing rats, minute ventilation, respiratory frequency, and neuromuscular inspiratory drive were lower in dexmedetomidine group, which also presented hypercapnia, whereas tidal volume, inspiratory, expiratory, and total respiratory cycle times were higher in dexmedetomidine group compared to the PENTO group. During mechanical ventilation, respiratory mechanical parameters were similar in both groups. These findings were supported by the absence of histological changes. In conclusion, under the conditions studied, dexmedetomidine did not change respiratory mechanical parameters and lung histology, but induced ventilatory depression.

Effects of different nutritional support on lung mechanics and remodelling in undernourished rats

This study investigated the impact of three different oral nutritional support regimens on lung mechanics and remodelling in young undernourished Wistar rats. In the nutritionally deprived group, rats received one-third of their usual daily food consumption for 4 weeks. Undernourished rats were divided into three groups receiving a balanced, glutamine-supplemented, or long-chain triglyceride-supplemented diet for 4 weeks. In the two control groups, rats received food ad libitum for 4 (C4) or 8 weeks. Lung viscoelastic pressure and static elastance were higher in undernourished compared to C4 rats. After refeeding, lung mechanical data remained altered except for the glutamine-supplemented group. Undernutrition led to a reduced amount of elastic and collagen fibres in the alveolar septa. Elastic fibre content returned to control with balanced and glutamine-supplemented diets, but increased with long-chain triglyceride-supplemented diet. The amount of collagen fibre augmented independent of nutritional support. In conclusion, glutamine-supplemented diet is better at reducing morphofunctional changes than other diets after 4 weeks of refeeding.

Alternating ventilation in a rat model of increased abdominal pressure

During alternating ventilation (AV) one lung is inflating while the other is deflating. Considering the possible respiratory and hemodynamic advantages of AV, we investigated its effects during increased intra-abdominal pressure (IAP=10 mmHg). In Sprague-Dawley rats (n=6, 270-375g) the main bronchi were independently cannulated, and respiratory mechanics determined while animals underwent different ventilatory patterns: synchronic ventilation without increased IAP (SV-0), elevated IAP during SV (SV-10), and AV with elevated IAP (AV-10). Thirty-three other animals (SV-0, n=10; SV-10, n=11 and AV-10, n=12) were ventilated during 3h. Mean arterial pressure (MAP), and lung histology were assessed. Increased IAP resulted in significantly higher elastances (p<0.001), being AV-10 lower than SV-10 (p<0.020). SV-10 showed higher central venous pressure (p<0.003) than S-0; no change was observed in AV-10. Wet/dry lung weight ratio was lower in AV-10 than SV-10 (p=0.009). Application of AV reduced hemodynamic and lung impairments induced by increased IAP during SV.