Elevated Oxidative Stress and DNA Damage in Cortical Neurons of Chemotherapy Patients.

The unintended neurologic sequelae of chemotherapy contribute to significant patient morbidity. Chemotherapy-related cognitive impairment (CRCI) is observed in up to 80% of cancer patients treated with chemotherapy and involves multiple cognitive domains including executive functioning. The pathophysiology underlying CRCI and the neurotoxicity of chemotherapy is incompletely understood, but oxidative stress and DNA damage are highly plausible mechanisms based on preclinical data. Unfortunately, validating pathways relevant to CRCI in humans is limited by an absence of relevant neuropathologic studies of patient brain tissue. In the present study, we stained sections of frontal lobe autopsy tissue from cancer patients treated with chemotherapy (n = 15), cancer patients not treated with chemotherapy (n = 10), and patients without history of cancer (n = 10) for markers of oxidative stress (nitrotyrosine, 4-hydroxynonenal) and DNA damage (pH2AX, pATM). Cancer patients treated with chemotherapy had increased staining for markers of oxidative stress and DNA damage in frontal lobe cortical neurons compared to controls. We detected no statistically significant difference in oxidative stress and DNA damage by the duration between last administration of chemotherapy and death. The study highlights the potential relevance of oxidative stress and DNA damage in the pathophysiology of CRCI and the neurotoxicity of chemotherapy.

Metabolic Profiling Reveals Aggravated Non-Alcoholic Steatohepatitis in High-Fat High-Cholesterol Diet-Fed Apolipoprotein E-Deficient Mice Lacking Ron Receptor Signaling.

Non-alcoholic steatohepatitis (NASH) represents the progressive sub-disease of non-alcoholic fatty liver disease that causes chronic liver injury initiated and sustained by steatosis and necroinflammation. The Ron receptor is a tyrosine kinase of the Met proto-oncogene family that potentially has a beneficial role in adipose and liver-specific inflammatory responses, as well as glucose and lipid metabolism. Since its discovery two decades ago, the Ron receptor has been extensively investigated for its differential roles on inflammation and cancer. Previously, we showed that Ron expression on tissue-resident macrophages limits inflammatory macrophage activation and promotes a repair phenotype, which can retard the progression of NASH in a diet-induced mouse model. However, the metabolic consequences of Ron activation have not previously been investigated. Here, we explored the effects of Ron receptor activation on major metabolic pathways that underlie the development and progression of NASH. Mice lacking apolipoprotein E (ApoE KO) and double knockout (DKO) mice that lack ApoE and Ron were maintained on a high-fat high-cholesterol diet for 18 weeks. We observed that, in DKO mice, the loss of ligand-dependent Ron signaling aggravated key pathological features in steatohepatitis, including steatosis, inflammation, oxidation stress, and hepatocyte damage. Transcriptional programs positively regulating fatty acid (FA) synthesis and uptake were upregulated in the absence of Ron receptor signaling, whereas lipid disposal pathways were downregulated. Consistent with the deregulation of lipid metabolism pathways, the DKO animals exhibited increased accumulation of FAs in the liver and decreased level of bile acids. Altogether, ligand-dependent Ron receptor activation provides protection from the deregulation of major metabolic pathways that initiate and aggravate non-alcoholic steatohepatitis.

Insights into Macrophage Heterogeneity and Cytokine-Induced Neuroinflammation in Major Depressive Disorder.

Over 350 million individuals suffer from depression, a psychiatric illness classified as major depressive disorder (MDD) with symptoms that include a loss of interest or pleasure in life accompanied by depressed mood. The present understanding of major depressive disorder does not encompass a systematic characterization of the neurobiological processes that drive the behavioral physiology in patients diagnosed with major depressive disorder. Psychiatric illness is a complex intersection between genetics, physiology, immunology and environmental stress. The increased attention to the relevance of depression has led to new discoveries that highlight the biological significance of ‘neuroinflammation’ and immunity underlying a spectrum of psychiatric illnesses. The process of neuroinflammation involves sentinel immune cells in the central nervous system (CNS). The activation and polarization of microglia, CNS-resident macrophages, modulates the production and secretion of pro-inflammatory cytokines implicated in the etiology of major depressive disorder, and this phenomenon has been aptly titled the ‘macrophage theory of depression’. Of particular interest are three hallmark cytokines, IL-6, TNFα and IL-1β, which have been studied extensively in basic research, cell-receptor signaling and drug development. The field of inflammasome-mediated neuroinflammation is an emerging area of MDD research that is providing new cellular insight into how macrophages mechanistically support cytokine-associated neuropathology, particularly in the case of IL-1β-associated inflammation in MDD. With the increasing number of individuals identified with depression, a comprehensive understanding of macrophage-cytokine signaling pathways in the CNS in depression is necessary for developing effective anti-depressant therapeutics.

Neuroprotective Role of the Ron Receptor Tyrosine Kinase Underlying Central Nervous System Inflammation in Health and Disease.

Neurodegeneration is a critical problem in aging populations and is characterized by severe central nervous system (CNS) inflammation. Macrophages closely regulate inflammation in the CNS and periphery by taking on different activation states. The source of inflammation in many neurodegenerative diseases has been preliminarily linked to a decrease in the CNS M2 macrophage population and a subsequent increase in M1-mediated neuroinflammation. The Recepteur D'Origine Nantais (Ron) is a receptor tyrosine kinase expressed on tissue-resident macrophages including microglia. Activation of Ron by its ligand, macrophage-stimulating protein, attenuates obesity-mediated inflammation in the periphery. An in vivo deletion of the ligand binding domain of Ron (Ron-/-) promotes inflammatory (M1) and limits a reparative (M2) macrophage activation. However, whether or not this response influences CNS inflammation has not been determined. In this study, we demonstrate that in homeostasis Ron-/- mice developed an inflammatory CNS niche with increased tissue expression of M1-associated markers when compared to age-matched wild-type (WT) mice. Baseline metabolic analysis of CNS tissue indicates exacerbated levels of metabolic stress in Ron-/- CNS. In a disease model of multiple sclerosis, experimental autoimmune encephalomyelitis, Ron-/- mice exhibit higher disease severity when compared to WT mice associated with increased CNS tissue inflammation. In a model of diet-induced obesity (DIO), Ron-/- mice exhibit exacerbated CNS inflammation with decreased expression of the M2 marker Arginase-1 (Arg-1) and a robust increase in M1 markers compared to WT mice following 27 weeks of DIO. Collectively, these results illustrate that activation of Ron in the CNS could be a potential therapeutic approach to treating various grades of CNS inflammation underlying neurodegeneration.

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

Aerobic Endurance Training Does Not Protect Bone Against Poorly Controlled Type 1 Diabetes in Young Adult Rats.

Streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) decreases trabecular bone volume and bone strength in rodents. The current study investigated the potential protective effects of aerobic endurance training (AET) on bone in STZ-induced T1DM young adult rats. Sixty-four 8-week-old male Sprague-Dawley rats were randomly divided into 4 groups of 16: control non-T1DM sedentary (CS) and exercised (CX), T1DM sedentary (DS) and exercised (DX). Blood glucose was maintained at 9-15 mmol/L using subcutaneously implanted insulin pellets (Linplant, Linshin Canada, Inc.). AET was performed at ~75-85% VO2max for 1 h/day, 5 day/week for 10 weeks. Areal and volumetric bone mineral density (aBMD and vBMD; excised femur) were measured using dual-energy X-ray absorptiometry (DXA; QDR 4500A) and micro computed tomography (µCT; Aloka). Bone strength was tested using a 3-point bending test (Instron 5544 Load Frame). Two-way ANOVA was used to test for T1DM and exercise differences followed by Tukey's HSD tests for interaction effects; significance was set at P < 0.05. T1DM had lower body weight (18.0%), aBMD (8.6%), cortical vBMD (1.6%), trabecular vBMD (2.1%), maximum load at break (22.2%), and increased elastic modulus (11.3%) vs. control (P < 0.001). Exercise in T1DM further decreased body weight (4.7%) vs. sedentary (P = 0.043) and maximum extension during the bending test that demonstrated DX was increased (7.3%) vs. CX (P = 0.033). There were no other beneficial effects of exercise on bone. These results suggest that 10 weeks of AET in rats do not have protective effects on bone in the short term and that T1DM rats have compromised bone health.

The Ron Receptor Tyrosine Kinase Regulates Macrophage Heterogeneity and Plays a Protective Role in Diet-Induced Obesity, Atherosclerosis, and Hepatosteatosis.

Obesity is a chronic inflammatory disease mediated in large part by the activation of inflammatory macrophages. This chronic inflammation underlies a whole host of diseases including atherosclerosis, hepatic steatosis, insulin resistance, type 2 diabetes, and cancer, among others. Macrophages are generally classified as either inflammatory or alternatively activated. Some tissue-resident macrophages are derived from yolk sac erythromyeloid progenitors and fetal liver progenitors that seed tissues during embryogenesis and have the ability to repopulate through local proliferation. These macrophages tend to be anti-inflammatory in nature and are generally involved in tissue remodeling, repair, and homeostasis. Alternatively, during chronic inflammation induced by obesity, bone marrow monocyte-derived macrophages are recruited to inflamed tissues, where they produce proinflammatory cytokines and exacerbate inflammation. The extent to which these two populations of macrophages are plastic in their phenotype remains controversial. We have demonstrated previously that the Ron receptor tyrosine kinase is expressed on tissue-resident macrophages, where it limits inflammatory macrophage activation and promotes a repair phenotype. In this study, we demonstrate that Ron is expressed in a subpopulation of macrophages during chronic inflammation induced by obesity that exhibit a repair phenotype as determined by the expression of arginase 1. In addition, we demonstrate that the Ron receptor plays a protective role in the progression of diet-induced obesity, hepatosteatosis, and atherosclerosis. These results suggest that altering macrophage heterogeneity in vivo could have the potential to alleviate obesity-associated diseases.

The glucoregulatory response to high-intensity aerobic exercise following training in rats with insulin-treated type 1 diabetes mellitus.

An acute bout of exercise elicits a rapid, potentially deleterious, reduction in blood glucose in patients with type 1 diabetes mellitus (T1DM). In the current study, we examined whether a 10-week aerobic training program could alleviate the rapid exercise-associated reduction in blood glucose through changes in the glucoregulatory hormonal response or increased hepatic glycogen storage in an insulin-treated rat model of T1DM. Thirty-two male Sprague-Dawley rats were divided evenly into 4 groups: non-T1DM sedentary (C) (n = 8), non-T1DM exercised (CX) (n = 8), T1DM sedentary (D) (n = 8), and T1DM exercised (DX) (n = 8). Exercise training consisted of treadmill running for 5 days/week (1 h, 27 m/min, 6% grade) for 10 weeks. T1DM was induced by multiple streptozotocin injections (20 mg/kg) followed by implantation of subcutaneous insulin pellets. At week 1, an acute exercise bout led to a significant reduction in blood glucose in DX (p < 0.05), whereas CX exhibited an increase in blood glucose (p < 0.05). During acute exercise, serum epinephrine was increased in both DX and CX (p < 0.05), whereas serum glucagon was increased during recovery only in CX (p < 0.01). Following aerobic training in DX, the exercise-mediated reduction in blood glucose remained; however, serum glucagon increased to the same extent as in CX (p < 0.05). DX exhibited significantly less hepatic glycogen (p < 0.001) despite elevations in glycogenic proteins in the liver (p < 0.05). Elevated serum epinephrine and decreased hepatic adrenergic receptor expression were also evident in DX (p < 0.05). In summary, despite aerobic training in DX, abrupt blood glucose reductions and hepatic glycogen deficiencies were evident. These data suggest that sympathetic overactivity may contribute to deficiencies in hepatic glycogen storage.

High Intensity Aerobic Exercise Training Improves Deficits of Cardiovascular Autonomic Function in a Rat Model of Type 1 Diabetes Mellitus with Moderate Hyperglycemia.

Indices of cardiovascular autonomic neuropathy (CAN) in experimental models of Type 1 diabetes mellitus (T1DM) are often contrary to clinical data. Here, we investigated whether a relatable insulin-treated model of T1DM would induce deficits in cardiovascular (CV) autonomic function more reflective of clinical results and if exercise training could prevent those deficits. Sixty-four rats were divided into four groups: sedentary control (C), sedentary T1DM (D), control exercise (CX), or T1DM exercise (DX). Diabetes was induced via multiple low-dose injections of streptozotocin and blood glucose was maintained at moderate hyperglycemia (9-17 mM) through insulin supplementation. Exercise training consisted of daily treadmill running for 10 weeks. Compared to C, D had blunted baroreflex sensitivity, increased vascular sympathetic tone, increased serum neuropeptide Y (NPY), and decreased intrinsic heart rate. In contrast, DX differed from D in all measures of CAN (except NPY), including heart rate variability. These findings demonstrate that this T1DM model elicits deficits and exercise-mediated improvements to CV autonomic function which are reflective of clinical T1DM.

The relationship between blood pressure and sciatic nerve blood flow velocity in rats with insulin-treated experimental diabetes.

Peripheral nerve blood flow (NBF) does not autoregulate but, instead, responds passively to changes in mean arterial pressure (MAP). How this relationship is impacted by insulin-treated experimental diabetes (ITED) is unknown. We tested the hypothesis that ITED will reduce NBF across a range of MAP in Sprague Dawley rats. Following 10 weeks of control or ITED conditions, conscious MAP (tail-cuff) was measured, and under anaesthesia, the MAP (carotid artery catheter, pressure transducer) and NBF (Doppler ultrasound, 40 MHz) responses to sodium nitroprusside (60 µg/kg) and phenylephrine (12 µg/kg) infusion were recorded (regression equations for MAP vs NBF were created for each rodent). Thereafter, motor nerve conduction velocity (MNCV) and nerve vascularization (haematoxylin and eosin stain) were determined. Conscious MAP was higher and MNCV was lower in the ITED group (p < 0.01). In response to drug infusions, the ΔMAP and ΔNBF were similar between groups (p ≥ 0.18). Estimated conscious NBF (based on substituting conscious MAP values into each individual regression equation) was greater in the ITED group (p < 0.01). Sciatic nerve vascularization was similar between groups (p ≥ 0.50). In contrast to the hypothesis, NBF was not reduced across a range of MAP. In spite of increased estimated conscious NBF values, MNCV was reduced in rats with ITED.

Exercise training enhances insulin-stimulated nerve arterial vasodilation in rats with insulin-treated experimental diabetes.

Insulin stimulates nerve arterial vasodilation through a nitric oxide (NO) synthase (NOS) mechanism. Experimental diabetes reduces vasa nervorum NO reactivity. Studies investigating hyperglycemia and nerve arterial vasodilation typically omit insulin treatment and use sedentary rats resulting in severe hyperglycemia. We tested the hypotheses that 1) insulin-treated experimental diabetes and inactivity (DS rats) will attenuate insulin-mediated nerve arterial vasodilation, and 2) deficits in vasodilation in DS rats will be overcome by concurrent exercise training (DX rats; 75-85% VO2 max, 1 h/day, 5 days/wk, for 10 wk). The baseline index of vascular conductance values (VCi = nerve blood flow velocity/mean arterial blood pressure) were similar (P ≥ 0.68), but peak VCi and the area under the curve (AUCi) for the VCi during a euglycemic hyperinsulinemic clamp (EHC; 10 mU·kg(-1)·min(-1)) were lower in DS rats versus control sedentary (CS) rats and DX rats (P ≤ 0.01). Motor nerve conduction velocity (MNCV) was lower in DS rats versus CS rats and DX rats (P ≤ 0.01). When compared with DS rats, DX rats expressed greater nerve endothelial NOS (eNOS) protein content (P = 0.04). In a separate analysis, we examined the impact of diabetes in exercise-trained rats alone. When compared with exercise-trained control rats (CX), DX rats had a lower AUCi during the EHC, lower MNCV values, and lower sciatic nerve eNOS protein content (P ≤ 0.03). Therefore, vasa nervorum and motor nerve function are impaired in DS rats. Such deficits in rats with diabetes can be overcome by concurrent exercise training. However, in exercise-trained rats (CX and DX groups), moderate hyperglycemia lowers vasa nervorum and nerve function.

Ontogeny and polarization of macrophages in inflammation: blood monocytes versus tissue macrophages.

The explosion of new information in recent years on the origin of macrophages in the steady-state and in the context of inflammation has opened up numerous new avenues of investigation and possibilities for therapeutic intervention. In contrast to the classical model of macrophage development, it is clear that tissue-resident macrophages can develop from yolk sac-derived erythro-myeloid progenitors, fetal liver progenitors, and bone marrow-derived monocytes. Under both homeostatic conditions and in response to pathophysiological insult, the contribution of these distinct sources of macrophages varies significantly between tissues. Furthermore, while all of these populations of macrophages appear to be capable of adopting the polarized M1/M2 phenotypes, their respective contribution to inflammation, resolution of inflammation, and tissue repair remains poorly understood and is likely to be tissue- and disease-dependent. A better understanding of the ontology and polarization capacity of macrophages in homeostasis and disease will be essential for the development of novel therapies that target the inherent plasticity of macrophages in the treatment of acute and chronic inflammatory disease.

High-intensity endurance training results in faster vessel-specific rate of vasorelaxation in type 1 diabetic rats.

This study examined the effects of 6 weeks of moderate- (MD) and high-intensity endurance training (HD) and resistance training (RD) on the vasorelaxation responsiveness of the aorta, iliac, and femoral vessels in type 1 diabetic (D) rats. Vasorelaxation to acetylcholine was modeled as a mono-exponential function. A potential mediator of vasorelaxation, endothelial nitric oxide synthase (e-NOS) was determined by Western blots. Vessel lumen-to-wall ratios were calculated from H&E stains. The vasorelaxation time-constant (τ) (s) was smaller in control (C) (7.2 ± 3.7) compared to D (9.1 ± 4.4) and it was smaller in HD (5.4 ± 1.5) compared to C, D, RD (8.3 ± 3.7) and MD (8.7 ± 3.8) (p<0.05). The rate of vasorelaxation (% · s(-1)) was larger in HD (2.7 ± 1.2) compared to C (2.0 ± 1.2), D (2.0 ± 1.5), RD (2.0 ± 1.0), and MD (2.0 ± 1.2) (p<0.05). τ vasorelaxation was smaller in the femoral (6.9 ± 3.7) and iliac (6.9 ± 4.7) than the aorta (9.0 ± 5.0) (p<0.05). The rate of vasorelaxation was progressively larger from the femoral (3.1 ± 1.4) to the iliac (2.0 ± 0.9) and to the aorta (1.3 ± 0.5) (p<0.05). e-NOS content (% of positive control) was greater in HD (104 ± 90) compared to C (71 ± 64), D (85 ± 65), RD (69 ± 43), and MD (76 ± 44) (p<0.05). e-NOS normalized to lumen-to-wall ratio (% · mm(-1)) was larger in the femoral (11.7 ± 11.1) compared to the aorta (3.2 ± 1.9) (p<0.05). Although vasorelaxation responses were vessel-specific, high-intensity endurance training was the most effective exercise modality in restoring the diabetes-related loss of vascular responsiveness. Changes in the vasoresponsiveness seem to be endothelium-dependent as evidenced by the greater e-NOS content in HD and the greater normalized e-NOS content in the smaller vessels.