A Rat Model of Clinically Relevant Extracorporeal Circulation Develops Early Organ Dysfunctions.

In clinical practice, extracorporeal circulation (ECC) is associated with coagulopathy and inflammation, eventually leading to organ injuries without preventive systemic pharmacological treatment. Relevant models are needed to reproduce the pathophysiology observed in humans and preclinical tests. Rodent models are less expensive than large models but require adaptations and validated comparisons to clinics. This study aimed to develop a rat ECC model and to establish its clinical relevance. One hour of veno-arterial ECC or a sham procedure were achieved on mechanically ventilated rats after cannulations with a mean arterial pressure objective > 60 mmHg. Five hours post-surgery, the rats' behavior, plasmatic/blood biomarkers, and hemodynamics were measured. Blood biomarkers and transcriptomic changes were compared in 41 patients undergoing on-pump cardiac surgery. Five hours post-ECC, the rats presented hypotension, hyperlactatemia, and behavioral alterations. The same patterns of marker measurements (Lactate dehydrogenase, Creatinine kinase, ASAT, ALAT, and Troponin T) were observed in both rats and human patients. Transcriptome analyses showed similarity in both humans and rats in the biological processes involved in the ECC response. This new ECC rat model seems to resemble both ECC clinical procedures and the associated pathophysiology, but with early organ injury corresponding to a severe phenotype. Although the mechanisms at stake in the post-ECC pathophysiology of rats or humans need to be described, this new rat model appears to be a relevant and costless preclinical model of human ECC.

Sex disparities in COVID-19 outcomes of inpatients with diabetes: insights from the CORONADO study.

OBJECTIVE: Male sex is one of the determinants of severe coronavirus diseas-e-2019 (COVID-19). We aimed to characterize sex differences in severe outcomes in adults with diabetes hospitalized for COVID-19. METHODS: We performed a sex-stratified analysis of clinical and biological features and outcomes (i.e. invasive mechanical ventilation (IMV), death, intensive care unit (ICU) admission and home discharge at day 7 (D7) or day 28 (D28)) in 2380 patients with diabetes hospitalized for COVID-19 and included in the nationwide CORONADO observational study (NCT04324736). RESULTS: The study population was predominantly male (63.5%). After multiple adjustments, female sex was negatively associated with the primary outcome (IMV and/or death, OR: 0.66 (0.49-0.88)), death (OR: 0.49 (0.30-0.79)) and ICU admission (OR: 0.57 (0.43-0.77)) at D7 but only with ICU admission (OR: 0.58 (0.43-0.77)) at D28. Older age and a history of microvascular complications were predictors of death at D28 in both sexes, while chronic obstructive pulmonary disease (COPD) was predictive of death in women only. At admission, C-reactive protein (CRP), aspartate amino transferase (AST) and estimated glomerular filtration rate (eGFR), according to the CKD-EPI formula predicted death in both sexes. Lymphocytopenia was an independent predictor of death in women only, while thrombocytopenia and elevated plasma glucose concentration were predictors of death in men only. CONCLUSIONS: In patients with diabetes admitted for COVID-19, female sex was associated with lower incidence of early severe outcomes, but did not influence the overall in-hospital mortality, suggesting that diabetes mitigates the female protection from COVID-19 severity. Sex-associated biological determinants may be useful to optimize COVID-19 prevention and management in women and men.

O-GlcNAc stimulation: A new metabolic approach to treat septic shock.

Septic shock is a systemic inflammation associated with cell metabolism disorders and cardiovascular dysfunction. Increases in O-GlcNAcylation have shown beneficial cardiovascular effects in acute pathologies. We used two different rat models to evaluate the beneficial effects of O-GlcNAc stimulation at the early phase of septic shock. Rats received lipopolysaccharide (LPS) to induce endotoxemic shock or saline (control) and fluid resuscitation (R) with or without O-GlcNAc stimulation (NButGT-10 mg/kg) 1 hour after shock induction. For the second model, rats received cecal ligature and puncture (CLP) surgery and fluid therapy with or without NButGT. Cardiovascular function was evaluated and heart and blood samples were collected and analysed. NButGT treatment efficiently increased total O-GlcNAc without modification of HBP enzyme expression.Treatment improved circulating parameters and cardiovascular function in both models, and restored SERCA2a expression levels. NButGT treatment also reduced animal mortality. In this study, we demonstrate that in septic shock O-GlcNAc stimulation improves global animal and cardiovascular function outcomes associated with a restoration of SERCA2a levels. This pre-clinical study opens avenues for a potential therapy of early-stage septic shock.

PCSK9 Concentrations in Cerebrospinal Fluid Are Not Specifically Increased in Alzheimer's Disease.

The role of PCSK9 in Alzheimer's disease (AD) is controversial. We compared cerebrospinal fluid (CSF) PCSK9 concentrations in 36 AD and 31 non-AD patients. CSF PCSK9 levels did not differ between AD and non-AD groups (2.80 versus 2.62 ng/mL). However, PCSK9 CSF levels were increased in AD and non-AD patients with other neurodegenerative process (non-AD ND, n = 20) compared to patients without neurodegenerative disorders (non-ND, n = 11): 2.80 versus 2.30 (p < 0.005) and 2.83 versus 2.30 ng/mL (p = NS), respectively. CSF PCSK9 were positively correlated with AD biomarkers (Aß1-42, T-tau, and P-tau). PCSK9 concentrations in CSF are increased in neurodegenerative disorders rather than specifically in AD.

Secondary reactive oxygen species production after PDT during pulmonary tumor growth in sera of nude mice.

Photodynamic therapy (PDT), mediated by a sensitizer exposed to light to produce singlet oxygen ((1)O2), induces tumor responses varying from one person to another. Cancer growth induces oxidative stress at any step of its development from induction to treatment, which could also modify response to PDT. After the initial amount of (1)O2 delivered, secondary oxidative species (SOS) are also generated inducing additional damages. Using an in vitro assay we saw variations among mice strains concerning their serum capability to generate SOS after (1)O2 production. Nude mice had a higher capability to generate SOS as compared to the non mutated strain. Capability to generate SOS evolved during growth of orthotopically-grafted pulmonary cancers (A549), with either values corrected for hemolysis or not. Immediately after graft SOS production decreased, then increased again, reaching a plateau phase after 10 days which lasted for 20 days and finally increased steeply during the last phase of tumor growth, preceding cachexia and death. This profile differed profoundly from the one observed after heterotopic tumor grafts for which hemolysis induced artifacts masking important variations in SOS production. Our results demonstrate experimentally a relationship between the general health status of an individual, cancer progression and serum capability to generate SOS during PDT. These findings could explain some PDT failures as well as some unexpected successes on large tumors and should be taken into account when determining treatment parameters. They may also explain why different effects are observed on different experimental models with similar sensitizers.

Evidence for antioxidants consumption in the coronary blood of patients with an acute myocardial infarction.

Blood flow arrest and reperfusion during myocardial infarction (MI) cause myocyte and endothelium injury through oxidative stress and inflammation, both of which involve reactive oxygen species (ROS) and peroxides that consume antioxidant defenses. The aim of this study was to determine whether serum from the occluded coronary vessel has impaired anti-oxidative defenses as compared to serum from aortic blood or from the periphery of healthy controls. Forty-seven patients (44 men) were included for study. Inclusion criteria were chest pain, ST elevation, and cardiac troponin increase. A photoreaction producing a standardized amount of singlet oxygen ((1)O2), an excited form of oxygen, was performed in serum samples obtained during primary percutaneous coronary intervention (PCI). Immediately after laser light delivery to 5% sera containing 5 µg/mL rose bengal, dichloro-dihydro-fluorescein (DCFH) was added and its post-oxidation transformation into the fluorescent DCF, was recorded. At least 5 h after the start of symptoms, the mean secondary ROS production after (1)O2 delivery was increased in coronary sera (p < 0.001), but in aortic blood remained similar to that of healthy controls. The peak troponin value correlated with DCF fluorescence throughout the interval between symptoms onset and PCI. A high fluorescence was associated with a higher risk of MACE. These results show that oxidants secondary to (1)O2 are increased in occluded vessels during AMI in parallel to c-troponin, demonstrating that antioxidants are consumed. A O2 increase during reperfusion would thus extend the damage resulting from IDM necrosis. The effect of conditioning during PCI could be studied using the described method.

Increased singlet oxygen-induced secondary ROS production in the serum of cancer patients.

Photodynamic therapy (PDT) generates singlet oxygen ((1)O(2)) and Reactive Oxygen Species (ROS) that are counteracted by patient's defenses. As cancer treatments are among the most important PDT applications the aim of this pilot study was to determine whether the serum of cancer patients produces more or less secondary ROS or peroxides after a photoreaction as compared to healthy persons. Fifty-three volunteers and 105 cancer patients were recruited. The capacity of (1)O(2) or secondary oxidant production was found to be increased in 6 healthy donors and 36 cancer patients (23/69 women and 13/31 men p<0.007 and p<0.04) with a mean value of 1.52 as compared to 1.29 in the healthy subjects (p<0.05) when considering values higher than the normal range (norm=1±10%) or 1.1 vs. 0.85 (p<0.01) in the whole cohort. This increase correlated with a poor prognosis, TNM and SBR classification. Serum (1)O(2) deactivation capacity was impaired and secondary ROS were more produced during cancer progression. Although it is currently unclear whether this is the cause or effect of cancer, this finding may hold interest as a potential marker of cancer severity. It would also support the interest of PDT as an adjuvant for cancer treatment, even for aggressive tumors particularly when associated to surgery for bulk removal.

Influence of animal species on secondary oxidant activation in serum exposed to singlet oxygen.

Singlet oxygen ((1)O(2)) produced during inflammatory reactions and during photodynamic therapy deactivates by producing in tissues secondary reactive oxygen species and peroxides (SOS) as well as other degradation products. We investigated the influence of animal species on SOS production secondary to standardized (1)O(2) production by performing in vitro experiments with rose bengal as the (1)O(2) producer, human serum (HS) as a control, sera derived from various animal species, and dichloro-dihydro-fluorescein as a nonspecific marker that becomes fluorescent when oxidized. The overall SOS production in HS from a presumed healthy cohort of 53 donors (31 males and 22 females) gave a mean "normal" value of 0.91 compared with a previous pool of 75 male sera samples. SOS production after a photo-reaction was two or four times lower in HS than in fetal calf serum or mice sera, respectively. In mice, the "nude" characteristic increased even more than in the SOS production. In the Aves order, this production appeared to be distributed randomly according to the number of branches after the appearance of Amniotas. For primates, SOS production appeared to decrease linearly with the number of branches (R(2) = 0.98). Adding hemolysates from complete bloods to the corresponding sera induced an increase in SOS production in all species, proportional to the production in sera. These findings should be kept in mind when interpreting results from studies of secondary reactive oxygen species-induced pathways following (1)O(2) production, regardless of its origin.

Serum resistance to singlet oxygen in patients with diabetes mellitus in comparison to healthy donors.

Diabetes mellitus causes endothelial injury through oxidative stress involving reactive oxygen species and peroxides as well as inflammation, both of which consume antioxidant defenses. Singlet oxygen ((1)O(2)) is produced by leukocytes during inflammatory and biochemical reactions and deactivated by producing reactive oxygen species and peroxides. To determine whether serum was capable of deactivating (1)O(2), we triggered a photo reaction in sera from 53 healthy donors and 52 diabetic patients. Immediately after light delivery, dichlorofluorescein was added and then its fluorescence was recorded. The mean capacity of (1)O(2) or secondary oxidant deactivation was reduced in patients with diabetes mellitus. Hemolysis reduced deactivation of (1)O(2)-induced secondary oxidants in both healthy and diabetic patients. Body mass index, age, platelet counts, and blood cell numbers exerted a nonlinear influence. High levels of glycated hemoglobin were associated with an increased deactivation of oxidative species, whereas high-density lipoprotein cholesterol, total cholesterol, and the total cholesterol to high-density lipoprotein cholesterol ratio decreased the serum deactivation capacity. Oral antidiabetics bore no influence on deactivation, which was restored by insulin in women. Deactivation capacity was lower in women, who had half the complications found in men, suggesting that, with more severe diabetes mellitus, protection was maintained against complications. Resistance to (1)O(2) should be considered during the monitoring of diabetes mellitus.

Biophysical parameters influencing secondary oxidants activation in human serum exposed to singlet oxygen.

Singlet oxygen (¹O2), produced during photodynamic therapy, deactivates during its interaction with tissues by producing reactive oxygen species (ROS) and peroxides as well as other degradation products. Here we investigated the role of parameters of light delivery, O(2), and temperature on the ROS and peroxides production, secondary to ¹O2. A series of simple in vitro experiments has been performed with Rose Bengal (RB) as a ¹O2 producer, human serum (HS) as a target and dichlorofluorescein (DCFH) as a nonspecific marker, becoming fluorescent when oxidized. The overall secondary production of ROS and peroxides in HS had also been compared to fetal calf serum (FCS) or mice sera. Increasing power but with a same delivered energy decreased secondary ROS and peroxides when increasing power with a same duration for light delivery increased them. Increasing delivered energy increased linearly secondary ROS or peroxides. Delivering O2 by bubbling before light delivery increased secondary ROS or peroxides, when Ar decreased them. Delivering gases after light delivery had no influence on secondary ROS or peroxides production. Increasing temperature from 20 to 40 °C increased secondary ROS or peroxides production but freezing after light delivery either before or after measurement had only a mild influence. Secondary ROS or peroxides production was 2 or 4 times lower in HS than in FCS or nude mice sera respectively. PDT seems to consist of two subsequent phases, both linked but developing independently. The intensity of photo-reactions varied with the model, human sera producing less secondary ROS than fetal calf or mouse sera. Search for new sensitizers should consider secondary ROS-induced pathways in addition to ¹O2 production.

Secondary oxidants in human serum exposed to singlet oxygen: the influence of hemolysis.

Singlet oxygen (1O2) is produced by leucocytes during inflammatory reactions, various biochemical reactions and during photoreactions. It deactivates by reacting with a number of targets to produce reactive oxygen species (ROS) and peroxides (that in turn produce ROS). To verify whether serum had the same capability to deactivate secondary oxidants after exposure to 1O2, we provoked a photoreaction using rose bengal added to sera of 53 healthy donors and, after light delivery, reduced 2',7'-dichlorofluorescein (DCFH) was added at the end of irradiation and fluorescence of the oxidized derivative (DCF) was recorded. To avoid optical artifacts, we analyzed the influence of hemolysis. Deactivation capability of secondary oxidants after exposure to (1)O(2) was stable over a long period of time, slightly different between men and women, but standard biochemistry parameters had little influence. Hemolysis, age and platelet number reduced deactivation of 1O2-induced secondary oxidants. Addition of lysed cancer cells had no influence. Blood sampling in clot act tubes gave a better signal than in heparinized tubes. Red blood cells (RBCs) loaded with antioxidants strongly decreased deactivation of secondary oxidants. Assays are in progress to evaluate the clinical implications of these findings.