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OBJECTIVE: To evaluate the use of the caudal vena cava collapsibility index (CVCCI) and the inspiratory/minimum and expiratory/maximum diameters of the vena cava to predict fluid responsiveness in hospitalized, critically ill cats with hemodynamic and tissue perfusion abnormalities. DESIGN: Diagnostic test study in a prospective cohort of hospitalized cats. SETTING: Private practice referral hospital. ANIMALS: Twenty-four hospitalized cats with spontaneous breathing and compromised hemodynamics and tissue hypoperfusion. INTERVENTIONS: Ultrasonographic examination before and after fluid expansion with 10 ml/kg of lactated Ringer's solution. MEASUREMENTS AND MAIN RESULTS: Fluid responsiveness was evaluated using the velocity-time integral (VTI) of the subaortic blood flow, by measuring it before and after a fluid load of 10 ml/kg of lactated Ringer's solution. The CVCCI was calculated using the following formula: (maximum diameter - minimum diameter / maximum diameter) × 100. Ten cats were fluid responders (42 %) and 14 were nonresponders (58 %). The area under the receiver operating characteristic curve (AUROC) with their 95% confidence interval for the predictors and the best cutoff values were as follows: CVCCI, AUROC = 0.83 (0.66-1.00) and cutoff = 31%; inspiratory/minimum diameter, AUROC = 0.86 (0.70-1.00) and cutoff = 0.24 cm; expiratory/maximum diameter, AUROC = 0.88 (0.74-1.00) and cutoff = 0.22 cm. A significant lineal correlation was observed between the percentage of increase in VTI after expansion and CVCCI (rs  = 0.68, P < 0.001), expiratory/maximum diameter (rs  = -0.72, P < 0.001), and inspiratory/minimum diameter (rs  = -0.71, P < 0.001). The intraobserver and interobserver variability was low for VTI, and the expiratory/maximum diameter and inspiratory/minimum diameter were high for CVCCI. CONCLUSIONS: Caudal vena cava measurements could be useful to predict the response to fluids in hospitalized cats with hemodynamic and tissue perfusion alterations. Additional studies are required to draw definitive conclusions about the role of these variables to guide fluid administration in cats.

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OBJECTIVE: To evaluate clinicopathological variables associated with hospital mortality in critically ill cats with compromised hemodynamics and tissue hypoperfusion. DESIGN: Retrospective observational study. SETTING: Private referral center. ANIMALS: Fifty-seven critically ill cats with compromised hemodynamics or tissue hypoperfusion. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The electronic medical records were searched for all cats admitted from June 2014 to November 2020. Cats were included in the study if the medical records clearly identified the presence of compromised hemodynamics and tissue hypoperfusion. Blood samples were obtained by percutaneous puncture of the external jugular vein, and blood gases, electrolytes, L-lactate concentration, and glucose were measured by a point-of-care analyzer. A predictive logistic regression model for mortality was performed. A total of 57 cats were ultimately included in the study. Thirty-five cats died. Eighteen of them were euthanized because of the severity of illness, and 17 died naturally. Twenty-two cats were discharged alive from the hospital. After adjusting for the Acute Patient Physiologic and Laboratory Evaluation (APPLE) fast score and disease category, jugular venous partial pressure of oxygen (Pvjo2 ) and HCT at admission were independent predictors of hospital mortality (HCT: odds ratio [OR], 0.763, 95% confidence interval [CI]: 0.625-0.930; P = 0.008; Pjvo2 : OR, 0.858; 95% CI: 0.749-0.984; P = 0.029). The association of these variables with mortality was maintained after conducting a sensitivity analysis and excluding cats that died by euthanasia. CONCLUSIONS: In cats with hemodynamic instability and tissue hypoperfusion, HCT and Pvjo2 behaved as independent predictors of mortality. Both variables seem to reflect the magnitude of oxygen debt and tissue hypoperfusion.

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BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe form of respiratory failure characterized by altered lung mechanics and poor oxygenation. Bronchial hyperresponsiveness has been reported in ARDS survivors and animal models of acute lung injury. Whether this hyperreactivity occurs at the small airways or not is unknown. OBJECTIVE: To determine ex-vivo small airway reactivity in a rat model of acute lung injury (ALI) by hydrochloric acid (HCl) instillation. METHODS: Twelve anesthetized rats were connected to mechanical ventilation for 4-hour, and randomly allocated to either ALI group (HCl intratracheal instillation; n=6) or Sham (intratracheal instillation of 0.9% NaCl; n=6). Oxygenation was assessed by arterial blood gases. After euthanasia, tissue samples from the right lung were harvested for histologic analysis and wet-dry weight ratio assessment. Precision cut lung slice technique (100-200 µm diameter) was applied in the left lung to evaluate ex vivo small airway constriction in response to histamine and carbachol stimulation, using phase-contrast video microscopy. RESULTS: Rats from the ALI group exhibited hypoxemia, worse histologic lung injury, and increased lung wet-dry weight ratio as compared with the sham group. The bronchoconstrictor responsiveness was significantly higher in the ALI group, both for carbachol (maximal contraction of 84.5±2.5% versus 61.4±4.2% in the Sham group, P<0.05), and for histamine (maximal contraction of 78.6±5.3% versus 49.6±5.3% in the Sham group, P<0.05). CONCLUSION: In an animal model of acute lung injury secondary to HCL instillation, small airway hyperresponsiveness to carbachol and histamine is present. These results may provide further insight into the pathophysiology of ARDS.

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INTRODUCTION: The use of antibiotics is mandatory in patients during extracorporeal membrane oxygenation (ECMO) support. Clinical studies have shown high variability in the antibiotic concentrations, as well as sequestration of them by the ECMO circuit, suggesting that the doses and/or interval administration used during ECMO may not be adequate. Thus, a fast response sensor to estimate antibiotic concentrations in this setting would contribute to improve dose adjustments. The biosensor PenP has been shown to have a dynamic range, sensitivity and specificity useful for pharmacokinetic (PK) tests in healthy subjects. However, the use of this biosensor in the context of a complex critical condition, such as ECMO during acute respiratory distress syndrome (ARDS), has not been tested. OBJECTIVES: To describe, by using PenP Biosensor, the pharmacokinetic of meropenem in a 24-h animal ARDS/ECMO model. METHODS: The PK of meropenem was evaluated in a swine model before and during ECMO. RESULTS: The PK parameters such as maximum concentration (Cmax), elimination rate constant (Ke), and cleareance (Cl), were not significantly altered during ECMO support. CONCLUSIONS: (a) ECMO does not affect the PK of meropenem, at least during the first 24 h; and (b) PenP has the potential to become an effective tool for making medical decisions associated with the dose model of antibiotics in a critical patient context.

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Insulin resistance is characteristic of pregnancies where the mother shows metabolic alterations, such as preeclampsia (PE) and gestational diabetes mellitus (GDM), or abnormal maternal conditions such as pregestational maternal obesity (PGMO). Insulin signalling includes activation of insulin receptor substrates 1 and 2 (IRS1/2) as well as Src homology 2 domain-containing transforming protein 1, leading to activation of 44 and 42 kDa mitogen-activated protein kinases and protein kinase B/Akt (Akt) signalling cascades in the human foetoplacental vasculature. PE, GDM, and PGMO are abnormal conditions coursing with reduced insulin signalling, but the possibility of the involvement of similar cell signalling mechanisms is not addressed. This review aimed to determine whether reduced insulin signalling in PE, GDM, and PGMO shares a common mechanism in the human foetoplacental vasculature. Insulin resistance in these pathological conditions results from reduced Akt activation mainly due to inhibition of IRS1/2, likely due to the increased activity of the mammalian target of rapamycin (mTOR) resulting from lower activity of adenosine monophosphate kinase. Thus, a defective signalling via Akt/mTOR in response to insulin is a central and common mechanism of insulin resistance in these diseases of pregnancy. In this review, we summarise the cell signalling mechanisms behind the insulin resistance state in PE, GDM, and PGMO focused in the Akt/mTOR signalling pathway in the human foetoplacental endothelium.

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Extracorporeal membrane oxygenation (ECMO) is increasingly being used to treat severe acute respiratory distress syndrome (ARDS). However, there is limited clinical evidence about how to optimize the technique. Experimental research can provide an alternative to fill the actual knowledge gap. The purpose of the present study was to develop and validate an animal model of acute lung injury (ALI) which resembled severe ARDS, and which could be successfully supported with ECMO. Eighteen pigs were randomly allocated into three groups: sham, ALI, and ALI + ECMO. ALI was induced by a double-hit consisting in repeated saline lavage followed by a 2-hour period of injurious ventilation. All animals were followed up to 24 hours while being ventilated with conventional ventilation (tidal volume 10 ml/kg). The lung injury model resulted in severe hypoxemia, increased airway pressures, pulmonary hypertension, and altered alveolar membrane barrier function, as indicated by an increased protein concentration in bronchoalveolar fluid, and increased wet/dry lung weight ratio. Histologic examination revealed severe diffuse alveolar damage, characteristic of ARDS. Veno-venous ECMO was started at the end of lung injury induction with a flow > 60 ml/kg/min resulting in rapid reversal of hypoxemia and pulmonary hypertension. Mortality was 0, 66.6 and 16.6% in the SHAM, ALI and ALI + ECMO groups, respectively (p < 0.05). This is a novel clinically relevant animal model that can be used to optimize the approach to ECMO and foster translational research in extracorporeal lung support.

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Nitric oxide plays several roles in cellular physiology, including control of the vascular tone and defence against pathogen infection. Neuronal, inducible and endothelial nitric oxide synthase (NOS) isoforms synthesize nitric oxide. Cells generate acid and base equivalents, whose physiological intracellular concentrations are kept due to membrane transport systems, including Na+ /H+ exchangers and Na+ /HCO3- transporters, thus maintaining a physiological pH at the intracellular (~7.0) and extracellular (~7.4) medium. In several pathologies, including cancer, cells are exposed to an extracellular acidic microenvironment, and the role for these membrane transport mechanisms in this phenomenon is likely. As altered NOS expression and activity is seen in cancer cells and because this gas promotes a glycolytic phenotype leading to extracellular acidosis in gynaecological cancer cells, a pro-inflammatory microenvironment increasing inducible NOS expression in this cell type is feasible. However, whether abnormal control of intracellular and extracellular pH by cancer cells regards with their ability to synthesize or respond to nitric oxide is unknown. We, here, discuss a potential link between pH alterations, pH controlling membrane transport systems and NOS function. We propose a potential association between inducible NOS induction and Na+ /H+ exchanger expression and activity in human ovary cancer. A potentiation between nitric oxide generation and the maintenance of a low extracellular pH (i.e. acidic) is proposed to establish a sequence of events in ovarian cancer cells, thus preserving a pro-proliferative acidic tumour extracellular microenvironment. We suggest that pharmacological therapeutic targeting of Na+ /H+ exchangers and inducible NOS may have benefits in human epithelial ovarian cancer.

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The placenta is a vital organ whose function in diseases of pregnancy is altered, resulting in an abnormal supply of nutrients to the foetus. The lack of placental vasculature homeostasis regulation causes endothelial dysfunction and altered vascular reactivity. The proper distribution of acid- (protons (H(+))) and base-equivalents through the placenta is essential to achieve physiological homeostasis. Several membrane transport mechanisms that control H(+) distribution between the extracellular and intracellular spaces are expressed in the human placenta vascular endothelium and syncytiotrophoblast, including sodium (Na(+))/H(+) exchangers (NHEs). One member of the NHEs family is NHE isoform 1 (NHE1), whose activity results in an alkaline intracellular pH (high intracellular pH (pHi)) and an acidic extracellular pH (pHo). Increased NHE1 expression, maximal transport activity, and turnover are reported in human syncytiotrophoblasts and lymphocytes from patients with diabetes mellitus type I (DMT1), and a positive correlation between NHEs activity and plasma factors, such as that between thrombin and platelet factor 3, has been reported in diabetes mellitus type II (DMT2). However, gestational diabetes mellitus (GDM) could result in a higher sensitivity of the human placenta to acidic pHo. We summarized the findings on pHi and pHo modulation in the human placenta with an emphasis on pregnancies in which the mother diagnosed with diabetes mellitus. A potential role of NHEs, particularly NHE1, is proposed regarding placental dysfunction in DMT1, DMT2, and GDM.

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The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 µmol/L STa (30 minutes), 25 µmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 µmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 µmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 µmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.

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Gastric aspiration is a high-risk condition for lung injury. Consequences range from subclinical pneumonitis to respiratory failure, with fibrosis development in some patients. Little is known about how the lung repairs aspiration-induced injury. By using a rat model of single orotracheal instillation of whole gastric contents, we studied the time course of morphological and biochemical changes during injury and resolution, and evaluated whether repair involved long-term fibrosis. Anesthetized rats received one gastric fluid instillation. At 4, 12, and 24 hours and 4 and 7 days, we performed lung histological studies and biochemical measurements in bronchoalveolar lavage fluid and lung tissue. Physiological measurements were performed at 12 to 24 hours. Long-term outcome was studied histologically at day 60. During the first 24 hours, severe peribronchiolar injury involving edema, intra-alveolar proteinaceous debris, hemorrhage, increased neutrophils and cytokines, and physiological dysfunction were observed. At days 4 and 7, an organizing pneumonia (OP) pattern developed, with foreign-body giant cells and granulomas. Lung matrix metalloproteinase 9 and 2 activities increased, with metalloproteinase-9 linked to early inflammation and metalloproteinase-2 to OP. At day 60, lung architecture was normal. In conclusion, a continuum of alterations starting with severe injury, evolving toward OP and later resolving, characterizes the rat single aspiration event. In addition to identifying markers of staging and severity, this model reveals that OP participates in the repair of aspiration-induced injury.

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