Peripheral and central changes induced by neural mobilization in animal models of neuropathic pain: a systematic review.

Introduction: Neural mobilization (NM) is a physiotherapy technique involving the passive mobilization of limb nerve structures with the aim to attempt to restore normal movement and structural properties. In recent years, human studies have shown pain relief in various neuropathic diseases and other pathologies as a result of this technique. Improvement in the range of motion (ROM), muscle strength and endurance, limb function, and postural control were considered beneficial effects of NM. To determine which systems generate these effects, it is necessary to conduct studies using animal models. The objective of this study was to gather information on the physiological effects of NM on the peripheral and central nervous systems (PNS and CNS) in animal models. Methods: The search was performed in Medline, Pubmed and Web of Science and included 8 studies according to the inclusion criteria. Results: The physiological effects found in the nervous system included the analgesic, particularly the endogenous opioid pathway, the inflammatory, by modulation of cytokines, and the immune system. Conclusion: On the basis of these results, we can conclude that NM physiologically modifies the peripheral and central nervous systems in animal models.

Identification of novel metabolomic biomarkers in an experimental model of septic acute kidney injury.

The aim of this study is the identification of metabolomic biomarkers of sepsis and sepsis-induced acute kidney injury (AKI) in an experimental model. Pigs were anesthetized and monitored to measure mean arterial pressure (MAP), systemic blood flow (QT), mean pulmonary arterial pressure, renal artery blood flow (QRA), renal cortical blood flow (QRC), and urine output (UO). Sepsis was induced at t = 0 min by the administration of live Escherichia coli ( n = 6) or saline ( n = 8). At t = 300 min, animals were killed. Renal tissue, urine, and serum samples were analyzed by nuclear magnetic resonance (NMR) spectroscopy. Principal component analyses were performed on the processed NMR spectra to highlight kidney injury biomarkers. Sepsis was associated with decreased QT and MAP and decreased QRA, QRC, and UO. Creatinine serum concentration and neutrophil gelatinase-associated lipocalin (NGAL) serum and urine concentrations increased. NMR-based metabolomics analysis found metabolic differences between control and septic animals: 1) in kidney tissue, increased lactate and nicotinuric acid and decreased valine, aspartate, glucose, and threonine; 2) in urine, increased isovaleroglycine, aminoadipic acid, N-acetylglutamine, N-acetylaspartate, and ascorbic acid and decreased myoinositol and phenylacetylglycine; and 3) in serum, increased lactate, alanine, pyruvate, and glutamine and decreased valine, glucose, and betaine concentrations. The concentration of several metabolites altered in renal tissue and urine samples from septic animals showed a significant correlation with markers of AKI (i.e., creatinine and NGAL serum concentrations). NMR-based metabolomics is a potentially useful tool for biomarker identification of sepsis-induced AKI.

Inhibition of Nitro-Oxidative Stress Attenuates Pulmonary and Systemic Injury Induced by High-Tidal Volume Mechanical Ventilation.

Mechanisms contributing to pulmonary and systemic injury induced by high tidal volume (VT) mechanical ventilation are not well known. We tested the hypothesis that increased peroxynitrite formation is involved in organ injury and dysfunction induced by mechanical ventilation. Male Sprague-Dawley rats were subject to low- (VT, 9 mL/kg; positive end-expiratory pressure, 5 cmH2O) or high- (VT, 25 mL/kg; positive end-expiratory pressure, 0 cmH2O) VT mechanical ventilation for 120 min, and received 1 of 3 treatments: 3-aminobenzamide (3-AB, 10 mg/kg, intravenous, a poly adenosine diphosphate ribose polymerase [PARP] inhibitor), or the metalloporphyrin manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 5 mg/kg intravenous, a peroxynitrite scavenger), or no treatment (control group), 30 min before starting the mechanical ventilation protocol (n = 8 per group, 6 treatment groups). We measured mean arterial pressure, peak inspiratory airway pressure, blood chemistry, and gas exchange. Oxidation (fluorescence for oxidized dihydroethidium), protein nitration (immunofluorescence and Western blot for 3-nitrotyrosine), PARP protein (Western blot) and gene expression of the nitric oxide (NO) synthase (NOS) isoforms (quantitative real-time reverse transcription polymerase chain reaction) were measured in lung and vascular tissue. Lung injury was quantified by light microscopy. High-VT mechanical ventilation was associated with hypotension, increased peak inspiratory airway pressure, worsened oxygenation; oxidation and protein nitration in lung and aortic tissue; increased PARP protein in lung; up-regulation of NOS isoforms in lung tissue; signs of diffuse alveolar damage at histological examination. Treatment with 3AB or MnTMPyP attenuated the high-VT mechanical ventilation-induced changes in pulmonary and cardiovascular function; down-regulated the expression of NOS1, NOS2, and NOS3; decreased oxidation and nitration in lung and aortic tissue; and attenuated histological changes. Increased peroxynitrite formation is involved in mechanical ventilation-induced pulmonary and vascular dysfunction.

Influence of mechanical ventilation and sepsis on redox balance in diaphragm, myocardium, limb muscles, and lungs.

Mechanical ventilation (MV), using high tidal volumes (V(T)), causes lung (ventilator-induced lung injury [VILI]) and distant organ injury. Additionally, sepsis is characterized by increased oxidative stress. We tested whether MV is associated with enhanced oxidative stress in sepsis, the commonest underlying condition in clinical acute lung injury. Protein carbonylation and nitration, antioxidants, and inflammation (immunoblotting) were evaluated in diaphragm, gastrocnemius, soleus, myocardium, and lungs of nonseptic and septic (cecal ligation and puncture 24 hours before MV) rats undergoing MV (n = 7 per group) for 150 minutes using 3 different strategies (low V(T) [V(T) = 9 mL/kg], moderate V(T) [V(T) = 15 mL/kg], and high V(T) [V(T) = 25 mL/kg]) and in nonventilated control animals. Compared with nonventilated control animals, in septic and nonseptic rodents (1) diaphragms, limb muscles, and myocardium of high-V(T) rats exhibited a decrease in protein oxidation and nitration levels, (2) antioxidant levels followed a specific fiber-type distribution in slow- and fast-twitch muscles, (3) tumor necrosis factor α (TNF-α) levels were higher in respiratory and limb muscles, whereas no differences were observed in myocardium, and (4) in lungs, protein oxidation was increased, antioxidants were rather decreased, and TNF-α remained unmodified. In this model of VILI, oxidative stress does not occur in distant organs or skeletal muscles of rodents after several hours of MV with moderate-to-high V(T), whereas protein oxidation levels were increased in the lungs of the animals. Inflammatory events were moderately expressed in skeletal muscles and lungs of the MV rats. Concomitant sepsis did not strongly affect the MV-induced effects on muscles, myocardium, or lungs in the rodents.

On the minute by minute variations of urine output: a study in a porcine model.

BACKGROUND: Urine output (UO) is usually measured hourly in acutely ill patients. Devices capable of more continuous (minute by minute urine output, UOm) measurements have become available recently. This paper aims to (1) analyze the minute by minute variations of UO, (2) analyze the impact of sepsis on those variations and (3) test if UO measured over periods shorter than 60 min provides information not available in hourly measurements. METHODS: Fifteen male pigs were anesthetized, tracheostomized and mechanically ventilated. Sepsis was induced by the administration of live Escherichia coli. Three groups were studied: nonseptic (n = 7) and septic (n = 4), both receiving sodium chloride (NaCl) 0.9 % at 4 ml kg(-1) h(-1); and septic (n = 4) receiving NaCl 0.9 % at 17 ml kg(-1) h(-1). UOm was measured during 6 h. RESULTS: There was a significant variation of UOm over time, as assessed by the coefficient of variation of the root-mean-squared error (CV(RMSE)), which was significantly more pronounced under conditions of sepsis than under control conditions. A UO production pattern in sepsis was identified, characterized by low UO production compared to baseline levels for approximately 30 min, followed by high UO production for approximately 30 min after initiation of the septic challenge. This pattern was noticeable if UO was measured every 10 min but not over longer periods of time. CONCLUSIONS: UOm provides information not conveyed by hourly measurements, especially under the cardiovascular alterations associated to sepsis. This information could enable an early identification of sepsis.

A Metabolomic Approach to the Pathogenesis of Ventilator-induced Lung Injury.

BACKGROUND: Global metabolic profiling using quantitative nuclear magnetic resonance spectroscopy (MRS) and mass spectrometry (MS) is useful for biomarker discovery. The objective of this study was to discover biomarkers of acute lung injury induced by mechanical ventilation (ventilator-induced lung injury [VILI]), by using MRS and MS. METHODS: Male Sprague-Dawley rats were subjected to two ventilatory strategies for 2.5 h: tidal volume 9 ml/kg, positive end-expiratory pressure 5 cm H2O (control, n = 14); and tidal volume 25 ml/kg and positive end-expiratory pressure 0 cm H2O (VILI, n = 10). Lung tissue, bronchoalveolar lavage fluid, and serum spectra were obtained by high-resolution magic angle spinning and H-MRS. Serum spectra were acquired by high-performance liquid chromatography coupled to quadupole-time of flight MS. Principal component and partial least squares analyses were performed. RESULTS: Metabolic profiling discriminated characteristics between control and VILI animals. As compared with the controls, animals with VILI showed by MRS higher concentrations of lactate and lower concentration of glucose and glycine in lung tissue, accompanied by increased levels of glucose, lactate, acetate, 3-hydroxybutyrate, and creatine in bronchoalveolar lavage fluid. In serum, increased levels of phosphatidylcholine, oleamide, sphinganine, hexadecenal and lysine, and decreased levels of lyso-phosphatidylcholine and sphingosine were identified by MS. CONCLUSIONS: This pilot study suggests that VILI is characterized by a particular metabolic profile that can be identified by MRS and MS. The metabolic profile, though preliminary and pending confirmation in larger data sets, suggests alterations in energy and membrane lipids.SUPPLEMENTAL DIGITAL CONTENT IS AVAILABLE IN THE TEXT.

Pulmonary vascular dysfunction induced by high tidal volume mechanical ventilation.

OBJECTIVES: Acute lung injury and acute respiratory distress syndrome are characterized by increased pulmonary artery pressure and ventilation-perfusion mismatch. We analyzed the changes in the pulmonary vascular function in a model of ventilator-induced acute lung injury. DESIGN: Controlled in vivo laboratory study. SETTING: Animal research laboratory. SUBJECTS: Anesthetized male Sprague-Dawley rats. INTERVENTIONS: Rats were ventilated for 120 minutes using low tidal volume ventilation (control group, tidal volume 9 mL/kg, positive end-expiratory pressure 5 cm H2O, n = 15), high tidal volume ventilation (high tidal volume group, tidal volume 25 mL/kg, zero positive end-expiratory pressure, n = 14), or high tidal volume ventilation plus the poly-(adenosine diphosphate-ribose) polymerase inhibitor 3-aminobenzamide (10 mg/kg IP, high tidal volume group + 3-aminobenzamide group, n = 7). Vascular rings from small pulmonary arteries were mounted in a myograph for isometric tension recording. Lung messenger RNA and protein expression were analyzed by reverse transcriptase-polymerase chain reaction and Western blot, respectively. MEASUREMENTS AND MAIN RESULTS: High tidal volume ventilation impaired phenylephrine- and acetylcholine-induced responses in pulmonary arteries in vitro, which were accompanied by induction of inducible nitric oxide synthase messenger RNA and protein. These effects, as well as hypoxemia and hypotension, were prevented by 3-aminobenzamide. Hypoxic pulmonary vasoconstriction and responses to exogenous sphingomyelinase were increased, whereas the responses to serotonin, Kv current density, and inhibition of Kv currents by hypoxia were unaffected by high tidal volume. CONCLUSIONS: High tidal volume ventilation-induced pulmonary vascular dysfunction was characterized by reduced alpha-adrenergic-induced vasoconstriction, reduced endothelium-dependent vasodilatation, and enhanced hypoxic pulmonary vasoconstriction.

Role of peroxynitrite in sepsis-induced acute kidney injury in an experimental model of sepsis in rats.

The mechanisms involved in sepsis-induced acute kidney injury (AKI) are unknown. We investigated the role of nitrosative stress in sepsis-induced AKI by studying the effects of manganese (III) tetrakis-(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), a peroxynitrite decomposition catalyst, and aminoguanidine (AG), a selective nitric oxide synthase 2 (NOS2) inhibitor and peroxynitrite scavenger, on kidney function of rats subjected to cecal ligation and puncture (CLP). Sprague-Dawley rats (weighing 350 [SD, 50] g) were treated with MnTMPyP (6 mg/kg i.p.) or AG (50 mg/kg i.p.) at t = 12 and 24 h after CLP or sham procedure. At t = 36 h, mean arterial pressure and aortic blood flow were measured, and blood and urine samples were obtained for biochemical determinations, including creatinine clearance, fractional excretion of sodium, and neutrophil gelatinase-associated lipocalin concentration in the urine. Kidney tissue samples were obtained for (i) light microscopy, (ii) immunofluorescence and Western blot for 3-nitrotyrosine and NOS2, (iii) gene expression (quantitative real-time polymerase chain reaction) studies (NOS1, NOS2, NOS3, and superoxide dismutase 1), and (iv) matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Mean arterial pressure was unchanged and aortic blood flow decreased 25% in CLP animals. The sepsis-induced (i) decreased urine output and creatinine clearance and increased fractional excretion of sodium and urinary neutrophil gelatinase-associated lipocalin concentration, (ii) increased protein nitration and NOS2 protein, and (iii) NOS1 and NOS2 upregulation were all significantly attenuated by treatment with MnTMPyP or AG. Nitrated proteins in renal tissue from CLP animals (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) were glutamate dehydrogenase, methylmalonate-semialdehyde dehydrogenase, and aldehyde dehydrogenase, mitochondrial proteins involved in energy metabolism or antioxidant defense. Nitro-oxidative stress is involved in sepsis-induced AKI, and protein nitration seems to be one mechanism involved.

A metabolomic approach for diagnosis of experimental sepsis.

BACKGROUND: The search for reliable diagnostic biomarkers of sepsis remains necessary. Assessment of global metabolic profiling using quantitative nuclear magnetic resonance (NMR)-based metabolomics offers an attractive modern methodology for fast and comprehensive determination of multiple circulating metabolites and for defining the metabolic phenotype of sepsis. OBJECTIVE: To develop a novel NMR-based metabolomic approach for diagnostic evaluation of sepsis. METHODS: Male Sprague-Dawley rats (weight 325-375 g) underwent cecal ligation and puncture (n = 14, septic group) or sham procedure (n = 14, control group) and 24 h later were euthanized. Lung tissue, bronchoalveolar lavage (BAL) fluid, and serum samples were obtained for (1)H NMR and high-resolution magic-angle spinning analysis. Unsupervised principal components analysis was performed on the processed spectra, and a predictive model for diagnosis of sepsis was constructed using partial least-squares discriminant analysis. RESULTS: NMR-based metabolic profiling discriminated characteristics between control and septic rats. Characteristic metabolites changed markedly in septic rats as compared with control rats: alanine, creatine, phosphoethanolamine, and myoinositol concentrations increased in lung tissue; creatine increased and myoinositol decreased in BAL fluid; and alanine, creatine, phosphoethanolamine, and acetoacetate increased whereas formate decreased in serum. A predictive model for diagnosis of sepsis using these metabolites classified cases with sensitivity and specificity of 100%. CONCLUSIONS: NMR metabolomic analysis is a potentially useful technique for diagnosis of sepsis. The concentrations of metabolites involved in energy metabolism and in the inflammatory response change in this model of sepsis.

Vascular dysfunction in sepsis: effects of the peroxynitrite decomposition catalyst MnTMPyP.

The mechanisms contributing to sepsis vascular dysfunction are not well known. We tested the hypothesis that peroxynitrite scavenging ameliorates sepsis-induced macrovascular and microvascular dysfunction. Male Sprague-Dawley rats were killed 48 h after cecal ligation (n = 15) and puncture or sham procedure (n = 15). Their aortas and mesenteric vessels were mounted in organ baths for isometric tension recording. We studied contraction in resting vessels (norepinephrine 1 nM-10 µM and 10 nM-10 µM) and endothelium-dependent relaxation (acetylcholine, 10 nM-10 µM and 1 nM-10 µM) for aortas and microvessels, respectively. Vascular rings were preincubated for 30 min with the superoxide scavenger Cu-Zn-superoxide dismutase (SOD) (100 U/mL), the SOD mimetic and peroxynitrite scavenger tempol (10 M), the NO synthase inhibitor N-nitro-l-arginine methyl ester (10 M), or the peroxynitrite decomposition catalyst manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP) (10 M). Fluorescence to 3-nitrotyrosine, oxidized dihydroethidium, and NOS2 was assessed in vascular tissue. Vascular NOS2, endothelial nitric oxide synthase (NOS1), NADPH-oxidase-1 (NOX-1), and SOD expression was analyzed by reverse transcription-polymerase chain reaction. Sepsis induced (i) in macrovessels, impairment of norepinephrine-induced contractions; (ii) in microvessels, impairment in norepinephrine-induced contractions and acetylcholine-induced relaxations; (iii) aortic and microvascular tissue increased reactivity to 3-nitrotyrosine, oxidized dihydroethidium, NOS2, and increased expression of NOS2, as well as increased expression of NOX-1 in microvascular tissue. Contractile responses in aortic and microvascular rings improved by ex vivo treatment with MnTMPyP and tempol, whereas vascular relaxation in microvessels improved only with MnTMPyP. Peroxynitrite scavenging protects from vascular dysfunction in sepsis.

Fluidizing effects of C-reactive protein on lung surfactant membranes: protective role of surfactant protein A.

The purpose of this study was to investigate how surfactant membranes can be perturbed by C-reactive protein (CRP) and whether surfactant protein A (SP-A) might overcome CRP-induced surfactant membrane alterations. The effect of CRP on surfactant surface adsorption was evaluated in vivo after intratracheal instillation of CRP into rat lungs. Insertion of CRP into surfactant membranes was investigated through monolayer techniques. The effect of CRP on membrane structure was studied through differential scanning calorimetry and fluorescence spectroscopy and microscopy using large and giant unilamellar vesicles. Our results indicate that CRP inserts into surfactant membranes and drastically increases membrane fluidity, resulting in surfactant inactivation. At 10% CRP/phospholipid weight ratio, CRP causes disappearance of liquid-ordered/liquid-disordered phase coexistence distinctive of surfactant membranes. SP-A, the most abundant surfactant lipoprotein structurally similar to C1q, binds to CRP (K(d)=56±8 nM), as determined by solid-phase binding assays and dynamic light scattering. This novel SP-A/CRP interaction reduces CRP insertion and blocks CRP effects on surfactant membranes. In addition, intratracheal coinstillation of SP-A+CRP into rat lungs prevents surfactant inhibition induced by CRP, indicating that SP-A/CRP interactions might be an important factor in vivo in controlling harmful CRP effects in the alveolus.

Redox balance and cellular inflammation in the diaphragm, limb muscles, and lungs of mechanically ventilated rats.

BACKGROUND: High tidal volume (VT) mechanical ventilation was shown to induce organ injury other than lung injury and systemic inflammation in animal models of ventilator-induced lung injury. The authors aimed to explore whether high VT mechanical ventilation per se induces early oxidative stress and inflammation in the diaphragm, limb muscles, and lungs of healthy rats exposed to ventilator-induced lung injury. METHODS: Protein carbonylation and nitration, antioxidants (immunoblotting), and inflammation (immunohistochemistry) were evaluated in the diaphragm, gastrocnemius, soleus, tibialis anterior, and lungs of mechanically ventilated healthy rats and in nonventilated control animals (n = 8/group) for 1 h, using two different strategies (moderate VT [VT = 9 ml/kg] and high VT [VT = 35 ml/kg]). RESULTS: The main findings are summarized as follows: compared with controls, (1) the diaphragms and gastrocnemius of high-VT rats exhibited a decrease in reactive carbonyls, (2) the soleus and tibialis of high- and moderate-VT rodents showed a reduction in reactive carbonyls and malondialdehyde-protein adducts, (3) the lungs of high-VT rats exhibited a significant rise in malondialdehyde-protein adducts, (4) the soleus and tibialis of both high- and moderate-VT rats showed a reduction in protein nitration, (5) the lungs of high- and moderate-VT rats showed a reduction in antioxidant enzyme levels, but not in the muscles, and (6) the diaphragms and gastrocnemius of all groups exhibited very low inflammatory cell counts, whereas the lungs of high-VT rats exhibited a significant increase in inflammatory infiltrates. CONCLUSIONS: Although oxidative stress and inflammation increased in the lungs of rats exposed to high VT, the diaphragm and limb muscles exhibited a decline in oxidative stress markers and very low levels of cellular inflammation.

Role of free radicals in vascular dysfunction induced by high tidal volume ventilation.

OBJECTIVE: To demonstrate that increased formation of reactive oxygen (ROS) and nitrogen species (RNS) is involved in VILI-induced vascular dysfunction. METHODS: Male Sprague-Dawley anesthetized rats were ventilated for 60 min using low V(T) ventilation [V(T) 9 ml/kg, positive end-expiratory pressure (PEEP) 5 cmH(2)O, n = 18], and high V(T) ventilation (V(T) 35 ml/kg, zero PEEP, n = 18). Arterial pressure and respiratory system mechanics were monitored. Blood samples for the determination of arterial blood gases and lactate concentration were drawn. Vascular rings from the thoracic aortae were mounted in organ baths for isometric tension recording. We studied endothelium-dependent relaxation in norepinephrine-precontracted rings (acetylcholine, 10 nM-10 microM) and contraction induced by norepinephrine (1 nM-10 microM) in resting vessels. Vascular rings were preincubated for 30 min with Zn-Mn-SOD (100 u/ml) or tempol (10(-4) M) (extracellular and intracellular superoxide scavengers, respectively) or MnTMPyP (10(-5) M) (a superoxide and peroxynitrite scavenger). The presence of superoxide and nitrotyrosine in aortic rings was evaluated by immunofluorescence. RESULTS: High V(T) ventilation induced hypotension, systemic acidosis, hypoxemia and hyperlactatemia, as well as impairment in acetylcholine and norepinephrine-induced responses in vitro. Responses to acetylcholine were improved by tempol (P = 0.004) and completely corrected (P < 0.001) by MnTMPyP. Responses to norepinephrine were also improved by treatment with tempol (P < 0.001) and MnTMPyP (P < 0.001). However, Zn-Mn-SOD did not improve acetylcholine- or norepinephrine-induced responses. Immunostaining for both superoxide and nitrotyrosine was increased in aortic rings from the high V(T) group. CONCLUSIONS: Our data support a role for intracellular ROS and peroxynitrite in the high V(T) ventilation-induced vascular dysfunction.

Aging increases the susceptibility to injurious mechanical ventilation.

OBJECTIVE: To test the hypothesis that aging increases the susceptibility to organ dysfunction and systemic inflammation induced by injurious mechanical ventilation. DESIGN AND SETTING: Experimental study in an animal model of ventilator-induced lung injury in the animal research laboratory in a university hospital. METHODS: Young (3-4 months old) and old (22-24 months old) anesthetized Wistar rats were ventilated for 60 min with a protective lung strategy (VT=9 ml/kg and PEEP=5 cm H2O, control) or with an injurious strategy (VT=35 ml/kg and PEEP=0 cm H2O, over-ventilated; n=6 for each group). MEASUREMENTS AND RESULTS: Mean arterial pressure and airway pressures (PAW) were monitored. Arterial blood gases and serum AST, ALT, lactate, and IL-6 were measured. Vascular rings from the thoracic aorta were mounted in organ baths for isometric tension recording. We studied relaxations induced by acetylcholine (10 nM-10 microM) in norepinephrine-precontracted rings, and contractions induced by norepinephrine (1 nM-10 microM) in resting vessels. Lungs were examined by light microscopy. Injurious ventilation in young rats was associated with hypoxemia, lactic metabolic acidosis, increased serum AST, hypotension, impairment in norepinephrine and acetylcholine-induced vascular responses ex vivo and hyaline membrane formation. The high-VT induced hypotension, increase in mean PAW, AST, and IL-6, and the impairment in acetylcholine-induced responses were significantly more marked in aged than in young rats. CONCLUSIONS: Elderly rats showed increased susceptibility to injurious mechanical ventilation-induced pulmonary injury, vascular dysfunction, and systemic inflammation.