A multimodal tissue perfusion measurement approach for the evaluation of the effect of pimobendan, an inodilator, in a porcine sepsis model.

Sepsis is associated with hypoperfusion and organ failure. The aims of the study were: 1) to assess the effect of pimobendan on macrocirculation and perfusion and 2) to describe a multimodal approach to the assessment of perfusion in sepsis and compare the evolution of the perfusion parameters. Eighteen anaesthetized female piglets were equipped for macrocirculation monitoring. Sepsis was induced by an infusion of Pseudomonas aeruginosa. After the occurrence of hypotension, animals were resuscitated. Nine pigs received pimobendan at the start of resuscitation maneuvers, the others received saline. Tissue perfusion was assessed using temperature gradients measured with infrared thermography (TG = core temperature - tarsus temperature), urethral perfusion index (uPI) derived from photoplethysmography and sublingual microcirculation (Sidestream dark field imaging device): De Backer score (DBs), proportion of perfused vessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI). Arterial lactate and ScvO2 were also measured. Pimobendan did not improve tissue perfusion nor macrocirculation. It did not allow a reduction in the amount of noradrenaline and fluids administered. Sepsis was associated with tissue perfusion disorders: there were a significant decrease in uPI, PPV and ScvO2 and a significant rise in TG. TG could significantly predict an increase in lactate. Resuscitation was associated with a significant increase in uPI, DBs, MFI, lactate and ScvO2. There were fair correlations between the different perfusion parameters. In this model, pimobendan did not show any benefit. The multimodal approach allowed the detection of tissue perfusion alteration but only temperature gradients predicted the increase in lactatemia.

Evaluation of stress markers in horses during hippotherapy sessions in comparison to being ridden by beginners.

Hippotherapy has been used for decades and its benefits to human patients have largely been proven, whether being applied to those with physical or mental disabilities. There have been a plethora of animal welfare studies recently, pertaining especially to ridden horses. This study aimed to investigate stress markers in horses during hippotherapy sessions to address the ethical considerations raised by using horses for therapy. A ridden stress ethogram was established and validated specifically for this study via subjective observation and video recording of a ridden session involving intermediate-level riders. The experiment entailed eight healthy horses undergoing two ridden sessions on separate days, one with disabled riders and one with beginners. Several parameters associated with physiological responses to stress were evaluated at rest, such as heart rate, plasma adrenocorticotropic hormone [ACTH], serum and salivary cortisol. These parameters as well as the behavioural stress score from the ethogram scale were measured during both sessions. No significant differences were found between heart rate, plasma ACTH, and stress scores. Serum and salivary cortisol were significantly lower during the hippotherapy session than during the session with beginners. The current study found no evidence of compromised welfare when horses were used as a therapeutic aid during hippotherapy sessions compared to their usual ridden activity. Although these results indicate that hippotherapy may be ethically justified as it benefits humans without causing harm to the horses, the present study was small, and the results should be interpreted with caution.

Feasibility study and direct extraction of endogenous free metallic cations combining hemodialysis and chelating polymer.

In this article, we report the conception and the use of dialysis-based medical device for the extraction of metals. The medical device is obtained by addition in the dialysate of a functionalized chitosan that can chelate endogenous metals like iron or copper. This water-soluble functionalized chitosan is obtained after controlled reacetylation and grafting of DOTAGA. Due to the high mass of chitosan, the polymer cannot cross through the membrane and the metals are trapped in the dialysate during hemodialysis. Copper extraction has been evaluated in vitro using an hemodialysis protocol. Feasibility study has been performed on healthy sheep showing no acute toxicity througout the entire dialysis procedure and first insights of metallic extraction even on healthy animals.

Microbial risk assessment of Nocardia cyriacigeorgica in polluted environments, case of urban rainfall water.

Urban Infiltration Basins (UIBs) are used to manage urban runoff transfers and feed aquifers. These UIBs can accumulate urban pollutants and favor the growth of potentially pathogenic biological agents as Nocardia. OBJECTIVES: To assess the spatio-temporal dynamics of pathogenic Nocardia in UIBs and to stablish phylogenetic relationships between clinical and UIB N. cyriacigeorgica strains. To assess pathogenicity associated with environmental N. cyriacigeorgica using an animal model, and to identify genetic elements that may be associated to its virulence. METHODS: A well-characterized UIB in terms of chemical pollutants from Lyon area was used in this study during a whole year. Cultural and Next-Generation-Sequencing methods were used for Nocardia detection and typing. Clinical and environmental isolates phylogenetic relationships and virulences were compared with Multilocus-Sequence-Analysis study together with a murine model. RESULTS: In autumn, N. cyriacigeorgica and N. nova were the pathogenic most prevalent species in the UIB. The complex N. abscessus/asiatica was also detected together with some other non-pathogenic species. The presence of pathogenic Nocardia was positively correlated to metallic trace elements. Up to 1.0 × 103 CFU/g sediment of N. cyriacigeorgica and 6 OTUs splited in two different phylogroups were retrieved and were close to clinical strains. The EML446 tested UIB isolate showed significant infectivity in mice with pulmonary damages similar to clinical clone (GUH-2). CONCLUSION: Hsp65 marker-based metabarcoding approach allowed detecting N. cyriacigeogica as the most abundant Nocardia pathogenic species in a UIB. Metal trace elements-polluted environments can be reservoirs of pathogenic Nocardia which may have a similar virulence to clinical strains.

Use of infrared thermography to detect early alterations of peripheral perfusion: evaluation in a porcine model.

This study aimed to evaluate the variations of infrared thermography according to rapid hemodynamic changes, by measuring the peripheral skin temperature in a porcine model. Eight healthy piglets were anesthetized and exposed to different levels of arterial pressure. Thermography was performed on the left forelimb to measure carpus and elbow skin temperature and their associated gradient with the core temperature. Changes in skin temperature in response to variations of blood pressure were observed. A negative correlation between arterial pressure and temperature gradients between peripheral and core temperature and a negative correlation between cardiac index and these temperature gradients were observed. Thermography may serve as a tool to detect early changes in peripheral perfusion.

PIPAC versus HIPEC: cisplatin spatial distribution and diffusion in a swine model.

Purpose: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a novel approach for delivering intraperitoneal chemotherapy and offers perspective in the treatment of peritoneal carcinomatosis. Concept is based on a 12 mmHg capnoperitoneum loaded with drug changed in microdoplets. It was postulated to guarantee a more homogeneous drug distribution and tissular uptake than hyperthermic intraperitoneal chemotherapy (HIPEC). The aim of this study was to compare cisplatin peritoneal distribution and pharmacokinetic between HIPEC and PIPAC procedures in a healthy swine model.Methods: Two groups of eight pigs underwent either HIPEC with cisplatin (70 mg/m2) at 43 °C for 60 min, or PIPAC with cisplatin (7.5 mg/m2) for 30 min. Postoperatively, peritoneal areas were biopsied allowing peritoneal cavity cartography. Tissular and plasmatic cisplatin concentrations were analyzed.Results: Cisplatin distribution was heterogeneous in both the groups with higher concentrations obtained closed to the delivery sites. Median total platinum peritoneal concentration by pig was higher in the HIPEC group than in the PIPAC group (18.0 µg/g versus 4.3 µg/g, p < .001) but the yield was 2.2 times better with PIPAC. Platinum concentrations were higher in the HIPEC group in all stations. At each time-point, cisplatin plasmatic concentrations were higher in the HIPEC group (p < .001) but beneath the toxicity threshold.Conclusions: With doses used in clinical practice, HIPEC guaranteed a higher cisplatin peritoneal uptake than PIPAC in this swine model. Spatial drug distribution was heterogeneous with both technics, with hotspots closed to the drug delivery sites. Nevertheless, considering the dose ratio, IP drug uptake yield was better with PIPAC.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) might increase the risk of anastomotic leakage compared to HIPEC: an experimental study.

BACKGROUND: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) and hyperthermic intraperitoneal chemotherapy (HIPEC) are technics proposed to treat patients with peritoneal carcinomatosis, in different settings. There is some concern about an over-risk of anastomotic leakage (AL) with PIPAC jeopardizing a combination with cytoreductive surgery. This study used a healthy swine model to compare the postoperative AL rate between PIPAC and HIPEC with digestive resection and to analyze macrocirculation and microcirculation parameters. METHODS: Segmental colonic resection with a handsewn anastomosis was performed on 16 healthy pigs; 8 pigs had a PIPAC procedure with 7.5 mg/m2 cisplatin (PIPAC group), and 8 pigs had a closed HIPEC procedure with 70 mg/m2 cisplatin and 42 °C as the target intraperitoneal temperature (HIPEC group). Pigs were kept alive for 8 days, then sacrificed and autopsied to look for AL, which was defined as local abscess or digestive fluid leakage when pressure was applied to the anastomosis. Food intake, weight, and core temperature were monitored postoperatively. Macrocirculation (heart rate, systolic blood pressure) and microcirculation parameters (percentage of perfused vessels, perfused vessels density, DeBacker score) were evaluated intraoperatively at five timepoints. Results were compared between pigs with AL and those without. RESULTS: The HIPEC group had no AL, but 3 of 8 pigs (37.5%) had AL in the PIPAC group (p = 0.20). Heart rate and core temperature showed perioperative increases in the HIPEC group. Intraoperatively, heart rate was higher in the HIPEC group at the two last timepoints (123 vs. 93 bpm, p = 0.031, and 110 vs. 85 bpm, p = 0.010, at timepoints 3 and 4, respectively). Other macrocirculatory and microcirculatory parameters showed no significant differences. CONCLUSION: In this healthy swine model, PIPAC might have increased AL incidence compared to HIPEC. This potential over-risk did not seem to be related to changes in the microcirculation. PIPAC should probably not be used with digestive resection and should be avoided in cases of perioperative serosal injury.

Genome Sequences of Three Nocardia cyriacigeorgica Strains and One Nocardia asteroides Strain.

We report four draft genome sequences of Nocardia spp. The strains are the Nocardia cyriacigeorgica DSM 44484 pathogenic type strain; two environmental isolates, Nocardia cyriacigeorgica EML446 and EML1456; and the Nocardia asteroides ATCC 19247 nonpathogenic type strain, with estimated genome sizes of 6.3 to 6.8 Mb. The study of these isolates will provide insight into physiology, evolution, and pathogenicity of Nocardia spp.

Mice Survival and Plasmatic Cytokine Secretion in a "Two Hit" Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load.

Sepsis is characterized by pro- and anti-inflammatory responses following infection. While inflammation is responsible for widespread organ damage, anti-inflammatory mediators lead to immunoparalysis increasing susceptibility to secondary infections (nosocomial pneumonia). We aimed to investigate the impact of bacterial load on survival and cytokine release in a two-hit murine (C57BL/6J) model of CLP followed by P. aeruginosa pneumonia. Plasmatic TNFα, IL-6, IL-10, sTNFr I and II were quantified until 13 days. At D5, splenocytes were processed for immunological assays or mice were intratracheally instilled with Pseudomonas aeruginosa (5.106, 2.107 and 108 CFU) to evaluate survival and cytokines production. TNFα, sTNFrs, IL-6 and IL-10 increased 2h post CLP. TNFα and sTNFrs declined respectively one and two days later. In CLP mice, IL-6 and IL-10 remained high for the whole experiment, as compared to Sham. At D5, for CLP mice, whereas total T cells population (CD3+) decreased, Treg fraction (CD4+/CD25+) increased. In parallel, T cells proliferation and LPS-stimulated splenocytes ability to release TNFα decreased. At D13, survival was 100% after 5.106 CFU, 50% for CLP mice after 2.107 CFU and 0% for CLP and Sham after 108 CFU. After instillation, IL-10 and IL-6 increased and appeared to be dose and time dependent. Pseudomonas was detected in all CLP and Sham's lungs; in spleen and liver only in CLP at 2.107 CFU, and in CLP and Sham at 108 CFU. We demonstrated that post-CLP immunosuppression followed by Pseudomonas aeruginosa lung instillation increases mortality reactivates cytokines secretion and is associated with systemic dissemination in septic mice depending on bacterial load.

Low WSS Induces Intimal Thickening, while Large WSS Variation and Inflammation Induce Medial Thinning, in an Animal Model of Atherosclerosis.

OBJECTIVE: Atherosclerotic plaque development in the arterial wall is the result of complex interaction between the wall's endothelial layer and blood hemodynamics. However, the interaction between hemodynamic parameters and inflammation in plaque evolution is not yet fully understood. The aim of the present study was to investigate the relation between wall shear stress (WSS) and vessel wall inflammation during atherosclerotic plaque development in a minipig model of carotid stenosis. METHODS: A surgical procedure was performed to create left common carotid artery stenosis by placement of a perivascular cuff in minipigs under atherogenic diet. Animals were followed up on 3T MRI, 1 week after surgery and 3, 6, and 8 months after initiation of the diet. Computational fluid dynamics simulation estimated WSS distribution for the first imaging point. Vascular geometries were co-registered for direct comparison of plaque development and features (Gadolinium- and USPIO-Contrast Enhanced MRI, for permeability and inflammation respectively) with the initial WSS. Histological analysis was performed and sections were matched to MR images, based on spatial landmarks. RESULTS: Vessel wall thickening, permeability and inflammation were observed distally from the stenosis. They were eccentric and facing regions of normal wall thickness. Histological analysis confirmed eccentric plaque formation with lipid infiltration, intimal thickening and medial degradation. High phagocytic activity in the stenosis region was co-localized with high WSS, corresponding to intense medial degradation observed on histology samples. CONCLUSION: Lower WSS promotes atherosclerotic plaque development distal to an induced stenosis. Vascular and perivascular inflammation locations were predominant in the high WSS stenosis segment, where medial thinning was the major consequence.

Inhibition of vascular smooth muscle cell proliferation and migration in vitro and neointimal hyperplasia in vivo by adenoviral-mediated atrial natriuretic peptide delivery.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation and migration are important components of the remodeling process in atherosclerosis or following angioplasty. Atrial natriuretic peptide (ANP) inhibits the growth of VSMCs in vitro but this effect has not been proven in vivo. In the present study, we examined the effects of local overexpression of ANP following gene transfer on in vitro VSMC proliferation and migration and in vivo neointimal formation in a rat carotid artery model of vascular injury. METHODS: ANP gene transfer was performed using a recombinant adenovirus containing the ANP cDNA controlled by the Rous sarcoma virus (RSV) long terminal repeat (Ad-RSV-ANP). A recombinant adenovirus expressing the RSV-controlled ß-galactosidase gene (Ad-RSV-ß-gal) was used as the control. Rat VSMC culture was used for in vitro studies. In the in vivo experiments, carotid arteries were analyzed after balloon injury and local infusion of the viral solution. RESULTS: VSMCs transfected by Ad-RSV-ANP produced a significant amount of ANP detected by immunoreactive assay and accumulated about 6.5 times more cGMP than the viral control. VSMC proliferation stimulated with 10% fetal calf serum was reduced by 31% and migration by 25%. Fourteen days after injury, neointimal formation and the intima/media ratio were reduced by 25% and 28%, respectively, in the Ad-RSV-ANP-treated group compared to the control group. CONCLUSIONS: The present study demonstrates the efficacy of recombinant adenovirus Ad-RSV-ANP with respect to inhibiting rat VSMC proliferation and migration. Our findings also provide evidence that ANP is implicated in the modulation of vascular remodeling following endothelial injury.

Pdx-1 or Pdx-1-VP16 protein transduction induces beta-cell gene expression in liver-stem WB cells.

BACKGROUND: Pancreatic duodenal homeobox-1 (Pdx-1) or Pdx-1-VP16 gene transfer has been shown to induce in vitro rat liver-stem WB cell conversion into pancreatic endocrine precursor cells. High glucose conditions were necessary for further differentiation into functional insulin-producing cells. Pdx-1 has the ability to permeate different cell types due to an inherent protein transduction domain (PTD). In this study, we evaluated liver-to-pancreas conversion of WB cells following Pdx-1 or Pdx-1-VP16 protein transduction. FINDINGS: WB cells were grown in high glucose medium containing Pdx-1 or Pdx-1-VP16 recombinant proteins for two weeks. beta-like cell commitment was analysed by RT-PCR of pancreatic endocrine genes. We found that WB cells in high glucose culture spontaneously express pancreatic endocrine genes (Pdx-1, Ngn3, Nkx2.2, Kir6.2). Their further differentiation into beta-like cells expressing genes related to endocrine pancreas development (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1, Pdx-1) and beta-cell function (Glut-2, Kir6.2, insulin) was achieved only in the presence of Pdx-1(-VP16) protein. CONCLUSION: These results demonstrate that Pdx-1(-VP16) protein transduction is instrumental for in vitro liver-to-pancreas conversion and is an alternative to gene therapy for beta-cell engineering for diabetes cell therapy.

Bipotential mouse embryonic liver (BMEL) cells spontaneously express Pdx1 and Ngn3 but do not undergo further pancreatic differentiation upon Hes1 down-regulation.

BACKGROUND: Liver-to-pancreas conversion offers new possibilities for beta-cell engineering for type 1 diabetes therapy. Among conceivable sources of liver cells, we focused on BMEL cells. These untransformed mouse embryonic liver cells have been reproducibly isolated from different inbred mice strains and have the potential to differentiate into hepatocytes and cholangiocytes in vitro and in vivo. FINDINGS: Strikingly, we find here that adherent BMEL cells display functional similarities with multipotent pancreatic precursor cells, namely Pdx1 and Ngn3 expression, and further express Hnf6 in floating aggregate culture. Hes1, a direct repressor of Ngn3 and pancreatic endocrine commitment, is expressed in adherent BMEL cells and decreases with time in aggregate culture. However, Hes1 decrease fails to initiate activation of late-stage pancreatic endocrine transcription factors. CONCLUSION: Here we report that BMEL cells present features of pancreatic endocrine progenitor cells. In the field of diabetes research, BMEL cells are of potential interest for the study of inductive signals critical for in vitro beta-cell maturation in-liver-to-pancreas conversion.

Adenovirus-mediated fibroblast growth factor 1 expression in the lung induces epithelial cell proliferation: consequences to hyperoxic lung injury in rats.

High concentrations of oxygen can induce pulmonary toxicity and cause injury to alveolar epithelial and endothelial cells. The present study was performed to determine whether the potent epithelial and endothelial fibroblast growth factor 1 (FGF-1) protected against hyperoxia-induced lung injury. Recombinant adenovirus carrying the gene encoding human secreted FGF-1 (Ad. FGF1) increased the proliferation of lung epithelial cells in vitro. Ad.FGF1 or control vector with an empty expression cassette (Ad.V152) was administered intratracheally to Wistar rats. With Ad.FGF1 (10(9), 5 x 10(9), 10(10), or 5 x 10(10) viral particles [VP]), FGF-1 protein was found in bronchoalveolar lavage fluid 4 days postinfection at levels proportional to the viral dose and was detected in plasma after doses of 10(10) VP or more were administered. Histological examination of the lungs showed intense proliferation and apoptosis of alveolar and bronchial epithelial cells, with few inflammatory cells. The alveolar architecture returned to normal within 17 days. Rats pretreated with Ad.FGF1 (10(9) or 5 x 10(9) VP) 2 days before exposure to hyperoxia (95% O2) survived, whereas rats pretreated with Ad.V152 died within 3 days. In conclusion, adenovirus-mediated FGF-1 overexpression in the lungs causes epithelial cell proliferation and has beneficial effects in hyperoxic lung injury.

Role of VEGF-B in the lung during development of chronic hypoxic pulmonary hypertension.

Angiogenic factors exert protective effects on the lung. To investigate the effect of VEGF-B, a factor coexpressed in the lung with VEGF-A, we assessed chronic hypoxic pulmonary hypertension in VEGF-B knockout mice (VEGF-B-/-) and in rats with lung overexpression of VEGF-B induced by adenovirus transfer. No significant difference in pulmonary hemodynamics, right ventricular hypertrophy, distal vessel muscularization, or vascular density was found between VEGF-B-/- and control mice after 3 wk of hypoxia. When overexpressed, VEGF-B(167) or VEGF-B(186) had protective effects similar to those of human VEGF-A(165). Lung endothelial nitric oxide synthase (eNOS) expression was increased by 5 days of hypoxia or VEGF-A adenovirus vector (Ad.VEGF-A) overexpression, whereas VEGF-B(167) or VEGF-B(186) had no effect. With hypoxia or normoxia, the wet-to-dry lung weight ratio was increased 5 days after Ad.VEGF-A administration compared with control (Ad.nul), Ad.VEGF-B(167), or Ad.VEGF-B(186). Endogenous VEGF-B does not counteract the development of hypoxic pulmonary hypertension. However, when overexpressed in the lung, VEGF-B can be as potent as VEGF-A in attenuating pulmonary hypertension, although it has no effect on eNOS expression or vascular permeability.