Developing a Clinically Relevant Hemorrhagic Shock Model in Rats.

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic shock, i.e., organ failure due to rapid loss of a large volume of blood, is associated with a highly complex pathophysiology involving several pathways. Numerous existing animal models of hemorrhagic shock strive to replicate what happens in humans, but these models have limits in terms of clinical relevance, reproducibility, or standardization. The aim of this study was to refine these models to develop a new model of hemorrhagic shock. Briefly, hemorrhagic shock was induced in male Wistar Han rats (11-13 weeks old) by a controlled exsanguination responsible for a drop in the mean arterial pressure. The next phase of 75 min was to maintain a low mean arterial blood pressure, between 32 mmHg and 38 mmHg, to trigger the pathophysiological pathways of hemorrhagic shock. The final phase of the protocol mimicked patient care with an administration of intravenous fluids, Ringer Lactate solution, to elevate the blood pressure. Lactate and behavioral scores were assessed 16 h after the protocol started, while hemodynamics parameters and plasmatic markers were evaluated 24 h after injury. Twenty-four hours post-hemorrhagic shock induction, the mean arterial and diastolic blood pressure were decreased in the hemorrhagic shock group (p < 0.05). Heart rate and systolic blood pressure remained unchanged. All organ damage markers were increased with the hemorrhagic shock (p < 0.05). The lactatemia and behavioral scores were increased compared to the sham group (p < 0.05). In conclusion, we demonstrated that the protocol described here is a relevant model of hemorrhagic shock that can be used in subsequent studies, particularly to evaluate the therapeutic potential of new molecules.

NButGT Reinforces the Beneficial Effects of Epinephrine on Cardiac Mitochondrial Respiration, Lactatemia and Cardiac Output in Experimental Anaphylactic Shock.

Anaphylactic shock (AS) is the most severe form of acute systemic hypersensitivity reaction. Although epinephrine can restore patients' hemodynamics, it might also be harmful, supporting the need for adjuvant treatment. We therefore investigated whether NButGT, enhancing O-GlcNAcylation and showing beneficial effects in acute heart failure might improve AS therapy. Ovalbumin-sensitized rats were randomly allocated to six groups: control (CON), shock (AS), shock treated with NButGT alone before (AS+pre-Nbut) or after (AS+post-Nbut) AS onset, shock treated with epinephrine alone (AS+EPI) and shock group treated with combination of epinephrine and NButGT (AS+EPI+preNBut). Induction of shock was performed with an intravenous (IV) ovalbumin. Cardiac protein and cycling enzymes O-GlcNAcylation levels, mean arterial pressure (MAP), heart rate, cardiac output (CO), left ventricle shortening fraction (LVSF), mitochondrial respiration, and lactatemia were evaluated using Western blotting experiments, invasive arterial monitoring, echocardiography, mitochondrial oximetry and arterial blood samples. AS decreased MAP (-77%, p < 0.001), CO (-90%, p < 0.001) and LVSF (-30%, p < 0.05). Epinephrine improved these parameters and, in particular, rats did not die in 15 min. But, cardiac mitochondrial respiration remained impaired (complexes I + II -29%, p < 0.05 and II -40%, p < 0.001) with hyperlactatemia. NButGT pretreatment (AS+pre-Nbut) efficiently increased cardiac O-GlcNAcylation level as compared to the AS+post-Nbut group. Compared to epinephrine alone, the adjunction of NButGT significantly improved CO, LVSF and mitochondrial respiration. MAP was not significantly increased but lactatemia decreased more markedly. Pretreatment with NButGT increases O-GlcNAcylation of cardiac proteins and has an additive effect on epinephrine, improving cardiac output and mitochondrial respiration and decreasing blood lactate levels. This new therapy might be useful when the risk of AS cannot be avoided.

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.

Spontaneous Sepsis in Adult Horses: From Veterinary to Human Medicine Perspectives.

Sepsis is a life-threatening disease defined as an organ dysfunction caused by a dysregulated host response to an infection. Early diagnosis and prognosis of sepsis are necessary for specific and timely treatment. However, no predictive biomarkers or therapeutic targets are available yet, mainly due to the lack of a pertinent model. A better understanding of the pathophysiological mechanisms associated with sepsis will allow for earlier and more appropriate management. For this purpose, experimental models of sepsis have been set up to decipher the progression and pathophysiology of human sepsis but also to identify new biomarkers or therapeutic targets. These experimental models, although imperfect, have mostly been performed on a murine model. However, due to the different pathophysiology of the species, the results obtained in these studies are difficult to transpose to humans. This underlines the importance of identifying pertinent situations to improve patient care. As humans, horses have the predisposition to develop sepsis spontaneously and may be a promising model for spontaneous sepsis. This review proposes to give first an overview of the different animal species used to model human sepsis, and, secondly, to focus on adult equine sepsis as a spontaneous model of sepsis and its potential implications for human and veterinary medicine.

An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights.

O-GlcNAcylation is a post-translational modification which affects approximately 5000 human proteins. Its involvement has been shown in many if not all biological processes. Variations in O-GlcNAcylation levels can be associated with the development of diseases. Deciphering the role of O-GlcNAcylation is an important issue to (i) understand its involvement in pathophysiological development and (ii) develop new therapeutic strategies to modulate O-GlcNAc levels. Over the past 30 years, despite the development of several approaches, knowledge of its role and regulation have remained limited. This review proposes an overview of the currently available tools to study O-GlcNAcylation and identify O-GlcNAcylated proteins. Briefly, we discuss pharmacological modulators, methods to study O-GlcNAcylation levels and approaches for O-GlcNAcylomic profiling. This review aims to contribute to a better understanding of the methods used to study O-GlcNAcylation and to promote efforts in the development of new strategies to explore this promising modification.

Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression.

The young population, which is particularly at risk of sepsis, is, paradoxically, rarely studied. Acute stimulation of O-GlcNAcylation, a post-translational modification involved in metabolic regulation, cell survival and stress response, is beneficial in young rats with sepsis. Considering that sepsis impacts the gene expression profile and that O-GlcNAcylation is a regulator of transcription, the aims of this study are to (i) unveil beneficial mechanisms of O-GlcNAcylation and (ii) decipher the relationship between O-GlcNAcylation and transcription during sepsis. Endotoxemic challenge was induced in 28-day-old male rats using a lipopolysaccharide injection (E. coli O111:B4, 20 mg·kg−1) and compared to control rats (NaCl 0.9%). One hour after, rats were assigned to no therapy or fluidotherapy (NaCl 0.9%, 10 mL.kg−1) ± NButGT (10 mg·kg−1) to stimulate O-GlcNAc levels. Cardiac O-GlcNAcylation levels were evaluated via Western blot and gene transcription using 3' SRP analysis. Lipopolysaccharide injection favorizes inflammatory state with the overexpression of genes involved in the NF-κB, JAK/STAT and MAPK pathways. NButGT treatment increased cardiac O-GlcNAcylation levels (p < 0.05). Yet, the mRNA expression was not impacted two hours after fluidotherapy or NButGT treatment. In conclusion, O-GlcNAc stimulation-induced beneficial effects are not dependent on the gene expression profile at the early phase of sepsis.

The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model.

50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of human ß 3-adrenoreceptor (Tgß 3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgß 3 rats. The aortic expression of • NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate • NO and O2 •- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgß 3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgß 3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21; Tgß 3: 6.08 ± 2.30; 45 weeks: WT: 1.00 ± 0.12; Tgß 3: 1.55 ± 0.17; p < 0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgß 3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37; Tgß 3: 0.13 ± 0.05; p < 0.05). Aortic •NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgß 3 rats (+52% vs. WT). Aortic O2 •- production was increased in Tgß 3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p < 0.05). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.

New Approaches to Identify Sepsis Biomarkers: The Importance of Model and Sample Source for Mass Spectrometry.

Septic shock is a systemic inflammatory response syndrome associated with circulatory failure leading to organ failure with a 40% mortality rate. Early diagnosis and prognosis of septic shock are necessary for specific and timely treatment. However, no predictive biomarker is available. In recent years, improvements in proteomics-based mass spectrometry have improved the detection of such biomarkers. This approach can be performed on different samples such as tissue or biological fluids. Working directly from human samples is complicated owing to interindividual variability. Indeed, patients are admitted at different stages of disease development and with signs of varying severity from one patient to another. All of these elements interfere with the identification of early, sensitive, and specific septic shock biomarkers. For these reasons, animal models of sepsis, although imperfect, are used to control the kinetics of the development of the pathology and to standardise experimentation, facilitating the identification of potential biomarkers. These elements underline the importance of the choice of animal model used and the sample to be studied during preclinical studies. The aim of this review is to discuss the relevance of different approaches to enable the identification of biomarkers that could indirectly be relevant to the clinical setting.