Early life stress exacerbates the obesogenic and anxiogenic effects of a Western diet without worsening cardiac ischaemic tolerance in male mice.

Early life stress (ELS) and a Western diet (WD) promote mood and cardiovascular disorders, however, how these risks interact in disease pathogenesis is unclear. We assessed effects of ELS with or without a subsequent WD on behaviour, cardiometabolic risk factors, and cardiac function/ischaemic tolerance in male mice. Fifty-six new-born male C57BL/6J mice were randomly allocated to a control group (CON) undisturbed before weaning, or to maternal separation (3h/day) and early (postnatal day 17) weaning (MSEW). Mice consumed standard rodent chow (CON, n = 14; MSEW, n = 15) or WD chow (WD, n = 19; MSEW + WD, n = 19) from week 8 to 24. Fasted blood was sampled and open field test and elevated plus maze (EPM) tests undertaken at 7, 15, and 23 weeks of age, with hearts excised at 24 weeks for Langendorff perfusion (evaluating pre- and post-ischaemic function). MSEW alone transiently increased open field activity at 7 weeks; body weight and serum triglycerides at 4 and 7 weeks, respectively; and final blood glucose levels and insulin resistance at 23 weeks. WD increased insulin resistance and body weight gain, the latter potentiated by MSEW. MSEW + WD was anxiogenic, reducing EPM open arm activity vs. WD alone. Although MSEW had modest metabolic effects and did not influence cardiac function or ischaemic tolerance in lean mice, it exacerbated weight gain and anxiogenesis, and improved ischaemic tolerance in WD fed animals. MSEW-induced increases in body weight (obesity) in WD fed animals in the absence of changes in insulin resistance may have protected the hearts of these mice.

Accelerated Hemocompatibility Testing of Rotary Blood Pumps.

Ex vivo hemocompatibility testing is a vital element of preclinical assessment for blood-contacting medical devices. Current approaches are resource intensive; thus, we investigated the feasibility of accelerating hemocompatibility testing by standardizing the number of pump exposures in loops of various sizes. Three identical blood loops were constructed, each with a custom-molded reservoir able to facilitate large-volume expansion. Using the HVAD rotary blood pump operating at 5 L·min -1 and 100 mmHg, three test volumes (80, 160, and 320 ml) were circulated for 4000 pump exposures. Blood sampling was performed at individualized intervals every one-sixth of total duration for the assessment of hemolysis and von Willebrand Factor (vWF) degradation. While steady increases in hemolysis (~24 mg·dl -1 ) were identified in all tests at completion, loop volume was not a primary discriminator. The normalized index of hemolysis did not vary significantly between loops (4.2-4.9 mg·100 L -1 ). vWF degradation progressively occurred with duration of testing to a similar extent under all conditions. These data support an accelerated approach to preclinical assessment of ex vivo blood damage. Adopting this approach enables: enhanced efficiency for rapid prototyping; reduced ex vivo blood aging, and; greater utility of blood, which is presently limited if 450 ml loops are desired.

Behavioural disruption in diabetic mice: Neurobiological correlates and influences of dietary α-linolenic acid.

AIM: Evolving type 2 diabetes (T2D) may influence locomotion and affective state, promoting metabolic dysfunction. We examined behaviour and neurobiology in a model of T2D, testing for benefits with dietary n-3 polyunsaturated fatty acid (PUFA). METHODS: Male C57Bl/6 mice received vehicle or 75 mg/kg streptozotocin (STZ) and 21 wks of control or Western diets (43 % fat, 40 % carbohydrate, 17 % protein). Sub-sets received dietary α-linolenic acid (ALA; 10 % of fat intake) for 6 wks. Behaviour was examined via open field and sucrose preference tests, and hippocampal and frontal cortex (FC) leptin and dopamine levels and inflammatory signalling assessed. KEY FINDINGS: T2D mice exhibited weight gain (+15 %), hyperglycemia (+35 %), hyperinsulinemia (+60 %) and insulin-resistance (+80 % higher HOMA-IR), together with anxiety-like behaviour (without anhedonia) that appeared independent of body weight and glycemic status. Cortical leptin declined whereas receptor mRNA increased. Supplementation with ALA did not influence metabolic state, while enhancing locomotion and reducing anxiety-like behaviours in healthy but not T2D mice. Hippocampal dopamine was selectively increased by ALA in T2D mice, with a trend to reduced circulating leptin in both groups. Across all groups, anxiety-like behaviour was associated with declining cortical and hippocampal leptin levels and increasing receptor mRNA, while declining dopamine levels were accompanied by decreased dopamine/serotonin receptor transcripts. SIGNIFICANCE: Chronic T2D induced anxiogenesis in mice appears to be independent of metabolic homeostasis but linked to central leptin-resistance, together with disturbed dopamine and serotonin signalling. Despite anxiolytic effects of ALA in healthy mice, no metabolic or behavioural benefits were evident in T2D.

A YOLO based software for automated detection and analysis of rodent behaviour in the open field arena.

Rodent models are important in mechanistic studies of the physiological and pathophysiological determinants of behaviour. The Open Field Test (OFT) is one of the most commonly utilised tests to assess rodent behaviour in a novel open environment. The key variables assessed in an OFT are general locomotor activity and exploratory behaviours and can be assessed manually or by automated systems. Although several automated systems exist, they are often expensive, difficult to use, or limited in the type of video that can be analysed. Here we describe a machine-learning algorithm - dubbed Cosevare - that uses a trained YOLOv3 DNN to identify and track movement of mice in the open-field arena. We validated Cosevare's capacity to accurately track locomotive and exploratory behaviour in 10 videos, comparing outputs generated by Cosevare with analysis by 5 manual scorers. Behavioural differences between control mice and those with diet-induced obesity (DIO) were also documented. We found the YOLOv3 based tracker to be accurate at identifying and tracking the mice within the open-field arena and in instances with variable backgrounds. Additionally, kinematic and spatial-based analysis demonstrated highly consistent scoring of locomotion, centre square duration (CSD) and entries (CSE) between Cosevare and manual scorers. Automated analysis was also able to distinguish behavioural differences between healthy control and DIO mice. The study found that a YOLOv3 based tracker is able to easily track mouse behaviour in the open field arena and supports machine learning as a potential future alternative for the assessment of animal behaviour in a wide range of species in differing environments and behavioural tests.

Dietary α-Linolenic Acid Counters Cardioprotective Dysfunction in Diabetic Mice: Unconventional PUFA Protection.

Whether dietary omega-3 (n-3) polyunsaturated fatty acid (PUFA) confers cardiac benefit in cardiometabolic disorders is unclear. We test whether dietary -linolenic acid (ALA) enhances myocardial resistance to ischemia-reperfusion (I-R) and responses to ischemic preconditioning (IPC) in type 2 diabetes (T2D); and involvement of conventional PUFA-dependent mechanisms (caveolins/cavins, kinase signaling, mitochondrial function, and inflammation). Eight-week male C57Bl/6 mice received streptozotocin (75 mg/kg) and 21 weeks high-fat/high-carbohydrate feeding. Half received ALA over six weeks. Responses to I-R/IPC were assessed in perfused hearts. Localization and expression of caveolins/cavins, protein kinase B (AKT), and glycogen synthase kinase-3 ß (GSK3ß); mitochondrial function; and inflammatory mediators were assessed. ALA reduced circulating leptin, without affecting body weight, glycemic dysfunction, or cholesterol. While I-R tolerance was unaltered, paradoxical injury with IPC was reversed to cardioprotection with ALA. However, post-ischemic apoptosis (nucleosome content) appeared unchanged. Benefit was not associated with shifts in localization or expression of caveolins/cavins, p-AKT, p-GSK3ß, or mitochondrial function. Despite mixed inflammatory mediator changes, tumor necrosis factor-a (TNF-a) was markedly reduced. Data collectively reveal a novel impact of ALA on cardioprotective dysfunction in T2D mice, unrelated to caveolins/cavins, mitochondrial, or stress kinase modulation. Although evidence suggests inflammatory involvement, the basis of this "un-conventional" protection remains to be identified.

Synergistic effects of low-level stress and a Western diet on metabolic homeostasis, mood, and myocardial ischemic tolerance.

How low-level psychological stress and overnutrition interact in influencing cardiometabolic disease is unclear. Mechanistic overlaps suggest potential synergies; however, findings are contradictory. We test whether low-level stress and Western diet (WD) feeding synergistically influence homeostasis, mood, and myocardial ischemic tolerance. Male C57BL6/J mice were fed a control diet or WD (32%/57%/11% calories from fat/carbohydrates/protein) for 12 wk, with subgroups restrained for 30 min/day over the final 3 wk. Metabolism, behavior, tolerance of perfused hearts to ischemia-reperfusion (I/R), and cardiac "death proteins" were assessed. The WD resulted in insignificant trends toward increased body weight (+5%), glucose (+40%), insulin (+40%), triglycerides (+15%), and cholesterol (+20%) and reduced leptin (-20%) while significantly reducing insulin sensitivity [100% rise in homeostasis model assessment of insulin resistance (HOMA-IR), P < 0.05]. Restraint did not independently influence metabolism while increasing HOMA-IR a further 50% (and resulting in significant elevations in insulin and glucose to 60-90% above control) in WD mice (P < 0.05), despite blunting weight gain in control and WD mice. Anxiogenesis with restraint or WD was nonadditive, whereas anhedonia (reduced sucrose consumption) only arose with their combination. Neuroinflammation markers (hippocampal TNF-α, Il-1b) were unchanged. Myocardial I/R tolerance was unaltered with stress or WD alone, whereas the combination worsened dysfunction and oncosis [lactate dehydrogenase (LDH) efflux]. Apoptosis (nucleosome accumulation) and death protein expression (BAK, BAX, BCL-2, RIP-1, TNF-α, cleaved caspase-3, and PARP) were unchanged. We conclude that mild, anxiogenic yet cardio-metabolically "benign" stress interacts synergistically with a WD to disrupt homeostasis, promote anhedonia (independently of neuroinflammation), and impair myocardial ischemic tolerance (independently of apoptosis and death protein levels).

Cardiomyoblast caveolin expression: Effects of simulated diabetes, α-linolenic acid and cell signaling pathways.

Caveolins regulate myocardial substrate handling, survival signaling and stress-resistance, however control of expression is incompletely defined. We test how metabolic features of type 2 diabetes (T2D), and modulation of cell signaling, influence caveolins in H9c2 cardiomyoblasts. Cells were exposed to glucose (25 vs. 5 mM), insulin (100 nM) or palmitate (0.1 mM), individually or combined, and effects of adenylate cyclase (AC) activation (50 µM forskolin), focal adhesion kinase (FAK) or protein kinase C b2 (PKCß2) inhibition (1 µM FAK Inhibitor 14 or CGP-53353, respectively), or the polyunsaturated fatty acid (PUFA) α-linolenic acid (ALA; 10 µM) were tested. Simulated T2D (elevated glucose+insulin+palmitate) depressed caveolin-1 and -3 without modifying caveolin-2. Caveolin-3 repression was primarily palmitate dependent, whereas high glucose (HG) and insulin independently increased caveolin-3 (yet reduced expression when combined). Differential control was evident: baseline caveolin-3 was suppressed by FAK/PKCß2 and insensitive to AC activities, with baseline caveolin-1 and -2 suppressed by AC and insensitive to FAK/PKCß2. Forskolin and ALA selectively preserved caveolin-3 in T2D cells, whereas PKCb2 and FAK inhibition increased caveolin-3 under all conditions. Despite preservation of caveolin-3, ALA did not modify nucleosome content (apoptosis marker) or transcription of pro-inflammatory mediators in T2D cells. In summary: caveolin-1 and -3 are strongly repressed with simulated T2D, with caveolin-3 particularly sensitive to palmitate; intrinsic PKCb2 and FAK activities repress caveolin-3 in healthy and stressed cells; ALA, AC activation and PKCß2 inhibition preserve caveolin-3 under T2D conditions; and caveolin-3 changes with T2D and ALA appear unrelated to inflammatory signaling and extent of apoptosis.

Chronic type 2 but not type 1 diabetes impairs myocardial ischaemic tolerance and preconditioning in C57Bl/6 mice.

NEW FINDINGS: • What is the central question of this study? What is the impact of chronic adult-onset diabetes on cardiac ischaemic outcomes and preconditioning? • What is the main finding and its importance? Chronic adult-onset type 2 but not type 1 diabetes significantly impairs myocardial ischaemic tolerance and ischaemic preconditioning. Preconditioning may be detrimental in type 2 diabetes, exaggerating nitrosative stress and apoptotic protein expression. ABSTRACT: Effects of diabetes on myocardial responses to ischaemia-reperfusion (I-R) and cardioprotective stimuli remain contentious, potentially reflecting influences of disease duration and time of onset. Chronic adult-onset type 1 diabetes (T1D) and type 2 diabetes (T2D) were modelled non-genetically in male C57Bl/6 mice via 5 × 50 mg kg-1 daily streptozotocin (STZ) injections + 12 weeks' standard chow or 1 × 75 mg kg-1 STZ injection + 12 weeks' obesogenic diet (32% calories as fat, 57% carbohydrate, 11% protein), respectively. Systemic outcomes were assessed and myocardial responses to I-R ± ischaemic preconditioning (IPC; 3 × 5 min I-R) determined in Langendorff perfused hearts. Uncontrolled T1D was characterised by pronounced hyperglycaemia (25 mm fasting glucose), glucose intolerance and ∼10% body weight loss, whereas T2D mice exhibited moderate hyperglycaemia (15 mm), hyperinsulinaemia, glucose intolerance and 17% weight gain. Circulating ghrelin, resistin and noradrenaline were unchanged with T1D, while leptin increased and noradrenaline declined in T2D mice. Ischaemic tolerance and IPC were preserved in T1D hearts. In contrast, T2D worsened post-ischaemic function (∼40% greater diastolic and contractile dysfunction) and cell death (100% higher troponin efflux), and abolished IPC protection. Whereas IPC reduced post-ischaemic nitrotyrosine and pro-apoptotic Bak and Bax levels in non-diabetic hearts, these effects were reduced in T1D and IPC augmented Bax and nitrosylation in T2D hearts. The data demonstrate chronic T1D does not inhibit myocardial I-R tolerance or IPC, whereas metabolic and endocrine disruption in T2D is associated with ischaemic intolerance and inhibition of IPC. Indeed, normally protective IPC may exaggerate damage mechanisms in T2D hearts.