Early Neuroprotective Effects of Bovine Lactoferrin Associated with Hypothermia after Neonatal Brain Hypoxia-Ischemia in Rats.

Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) in term newborns is a leading cause of mortality and chronic disability. Hypothermia (HT) is the only clinically available therapeutic intervention; however, its neuroprotective effects are limited. Lactoferrin (LF) is the major whey protein in milk presenting iron-binding, anti-inflammatory and anti-apoptotic properties and has been shown to protect very immature brains against HI damage. We hypothesized that combining early oral administration of LF with whole body hypothermia could enhance neuroprotection in a HIE rat model. Pregnant Wistar rats were fed an LF-supplemented diet (1 mg/kg) or a control diet from (P6). At P7, the male and female pups had the right common carotid artery occluded followed by hypoxia (8% O2 for 60') (HI). Immediately after hypoxia, hypothermia (target temperature of 32.5-33.5 °C) was performed (5 h duration) using Criticool®. The animals were divided according to diet, injury and thermal condition. At P8 (24 h after HI), the brain neurochemical profile was assessed using magnetic resonance spectroscopy (1H-MRS) and a hyperintense T2W signal was used to measure the brain lesions. The mRNA levels of the genes related to glutamatergic excitotoxicity, energy metabolism and inflammation were assessed in the right hippocampus. The cell markers and apoptosis expression were assessed using immunofluorescence in the right hippocampus. HI decreased the energy metabolites and increased lactate. The neuronal-astrocytic coupling impairments observed in the HI groups were reversed mainly by HT. LF had an important effect on astrocyte function, decreasing the levels of the genes related to glutamatergic excitotoxicity and restoring the mRNA levels of the genes related to metabolic support. When combined, LF and HT presented a synergistic effect and prevented lactate accumulation, decreased inflammation and reduced brain damage, pointing out the benefits of combining these therapies. Overall, we showed that through distinct mechanisms lactoferrin can enhance neuroprotection induced by HT following neonatal brain hypoxia-ischemia.

Altered heart cytokine profile and action potential modulation in cardiomyocytes from Mas-deficient mice.

The renin-angiotensin system (RAS) is a key hormonal system. In recent years, the functional analysis of the novel axis of the RAS (ACE2/Ang-(1-7)/Mas receptor) revealed that its activation can become protective against several pathologies, including cardiovascular diseases. Mas knockout mice (Mas-KO) represent an important tool for new investigations. Indeed, extensive biological research has focused on investigating the functional implications of Mas receptor deletion. However, although the Mas receptor was identified in neonatal cardiomyocytes and also in adult ventricular myocytes, only few reports have explored the Ang-(1-7)/Mas signaling directly in cardiomyocytes to date. This study investigated the implication of Mas receptor knockout to the cytokine profile, energy metabolism, and electrical properties of mice-isolated cardiomyocytes. Here, we demonstrated that Mas-KO mice have modulation in some cytokines, such as G-CSF, IL-6, IL-10, and VEGF in the left ventricle. This model also presents increased mitochondrial number in cardiomyocytes and a reduction in the myocyte diameter. Finally, Mas-KO cardiomyocytes have altered action potential modulation after diazoxide challenge. Such electrical finding was different from the data showed for the TGR(A1-7)3292 (TGR) model, which overexpresses Ang-(1-7) in the plasma by 4.5, used by us as a control. Collectively, our findings exemplify the importance of understanding the ACE2/Ang-(1-7)/Mas pathway in cardiomyocytes and heart tissue. The Mas-KO mice model can be considered an important tool for new RAS investigations.

Dose-Dependent Neuroprotective Effects of Bovine Lactoferrin Following Neonatal Hypoxia-Ischemia in the Immature Rat Brain.

Injuries to the developing brain due to hypoxia-ischemia (HI) are common causes of neurological disabilities in preterm babies. HI, with oxygen deprivation to the brain or reduced cerebral blood perfusion due to birth asphyxia, often leads to severe brain damage and sequelae. Injury mechanisms include glutamate excitotoxicity, oxidative stress, blood-brain barrier dysfunction, and exacerbated inflammation. Nutritional intervention is emerging as a therapeutic alternative to prevent and rescue brain from HI injury. Lactoferrin (Lf) is an iron-binding protein present in saliva, tears, and breast milk, which has been shown to have antioxidant, anti-inflammatory and anti-apoptotic properties when administered to mothers as a dietary supplement during pregnancy and/or lactation in preclinical studies of developmental brain injuries. However, despite Lf's promising neuroprotective effects, there is no established dose. Here, we tested three different doses of dietary maternal Lf supplementation using the postnatal day 3 HI model and evaluated the acute neurochemical damage profile using 1H Magnetic Resonance Spectroscopy (MRS) and long-term microstructure alterations using advanced diffusion imaging (DTI/NODDI) allied to protein expression and histological analysis. Pregnant Wistar rats were fed either control diet or bovine Lf supplemented chow at 0.1, 1, or 10 g/kg/body weight concentration from the last day of pregnancy (embryonic day 21-E21) to weaning. At postnatal day 3 (P3), pups from both sexes had their right common carotid artery permanently occluded and were exposed to 6% oxygen for 30 min. Sham rats had the incision but neither surgery nor hypoxia episode. At P4, MRS was performed on a 9.4 T scanner to obtain the neurochemical profile in the cortex. At P4 and P25, histological analysis and protein expression were assessed in the cortex and hippocampus. Brain volumes and ex vivo microstructural analysis using DTI/NODDI parameters were performed at P25. Acute metabolic disturbance induced in cortical tissue by HIP3 was reversed with all three doses of Lf. However, data obtained from MRS show that Lf neuroprotective effects were modulated by the dose. Through western blotting analysis, we observed that HI pups supplemented with Lf at 0.1 and 1 g/kg were able to counteract glutamatergic excitotoxicity and prevent metabolic failure. When 10 g/kg was administered, we observed reduced brain volumes, increased astrogliosis, and hypomyelination, pointing to detrimental effects of high Lf dose. In conclusion, Lf supplementation attenuates, in a dose-dependent manner, the acute and long-term cerebral injury caused by HI. Lf reached its optimal effects at a dose of 1 g/kg, which pinpoints the need to better understand effects of Lf, the pathways involved and possible harmful effects. These new data reinforce our knowledge regarding neuroprotection in developmental brain injury using Lf through lactation and provide new insights into lactoferrin's neuroprotection capacities and limitation for immature brains.

Synchronous nonmonotonic changes in functional connectivity and white matter integrity in a rat model of sporadic Alzheimer's disease.

Brain glucose hypometabolism has been singled out as an important contributor and possibly main trigger to Alzheimer's disease (AD). Intracerebroventricular injections of streptozotocin (icv-STZ) cause brain glucose hypometabolism without systemic diabetes. Here, a first-time longitudinal study of brain glucose metabolism, functional connectivity and white matter microstructure was performed in icv-STZ rats using PET and MRI. Histological markers of pathology were tested at an advanced stage of disease. STZ rats exhibited altered functional connectivity and intra-axonal damage and demyelination in brain regions typical of AD, in a temporal pattern of acute injury, transient recovery/compensation and chronic degeneration. In the context of sustained glucose hypometabolism, these nonmonotonic trends - also reported in behavioral studies of this animal model as well as in human AD - suggest a compensatory mechanism, possibly recruiting ketone bodies, that allows a partial and temporary repair of brain structure and function. The early acute phase could thus become a valuable therapeutic window to strengthen the recovery phase and prevent or delay chronic degeneration, to be considered both in preclinical and clinical studies of AD. In conclusion, this work reveals the consequences of brain insulin resistance on structure and function, highlights signature nonmonotonic trajectories in their evolution and proposes potent MRI-derived biomarkers translatable to human AD and diabetic populations.

Multi-slice passband bSSFP for human and rodent fMRI at ultra-high field.

Balanced steady-state free precession (bSSFP) can be used as an alternative to gradient-echo (GE) EPI for BOLD functional MRI when image distortions and signal drop-outs are severe such as at ultra-high field. However, 3D-bSSFP acquisitions have distinct drawbacks on either human or animal MR systems. On clinical scanners, 3D imaging is suboptimal for localized fMRI applications. It can also display distortions when acceleration methods such as spiral read-outs are used, and, compared to multi-slice acquisitions, suffers from increased sensitivity to motion or physiological noise which further results in blurring. On pre-clinical systems, 3D acquisitions have low temporal resolution due to limited acceleration options, while single slice often results in insufficient coverage. The aim of the present study was to implement a multi-slice bSSFP acquisition with Cartesian read-out to obtain non-distorted BOLD fMRI activation maps in the human and rat brain at ultra-high field. We show that, when using a new pseudo-steady-state, the bSSFP signal characteristics are preserved. In the human brain at 7 T, we demonstrate that both task- and resting-state fMRI can be performed with multi-slice bSSFP, with a temporal SNR that matches that of 3D-bSSFP, resulting in - at least - equal performance. In the rat brain at 14 T, we show that the multi-slice bSSFP protocol has similar sensitivity to gradient-echo EPI for task fMRI, while benefitting from much reduced distortions and drop-outs. The advantages of passband bSSFP at 14 T in comparison with GE-EPI are expected to be even more marked for mouse brain.

Brain Metabolism Alterations Induced by Pregnancy Swimming Decreases Neurological Impairments Following Neonatal Hypoxia-Ischemia in Very Immature Rats.

Introduction: Prematurity, through brain injury and altered development is a major cause of neurological impairments and can result in motor, cognitive and behavioral deficits later in life. Presently, there are no well-established effective therapies for preterm brain injury and the search for new strategies is needed. Intra-uterine environment plays a decisive role in brain maturation and interventions using the gestational window have been shown to influence long-term health in the offspring. In this study, we investigated whether pregnancy swimming can prevent the neurochemical metabolic alterations and damage that result from postnatal hypoxic-ischemic brain injury (HI) in very immature rats. Methods: Female pregnant Wistar rats were divided into swimming (SW) or sedentary (SE) groups. Following a period of adaptation before mating, swimming was performed during the entire gestation. At postnatal day (PND3), rat pups from SW and SE dams had right common carotid artery occluded, followed by systemic hypoxia. At PND4 (24 h after HI), the early neurochemical profile was measured by 1H-magnetic resonance spectroscopy. Astrogliosis, apoptosis and neurotrophins protein expression were assessed in the cortex and hippocampus. From PND45, behavioral testing was performed. Diffusion tensor imaging and neurite orientation dispersion and density imaging were used to evaluate brain microstructure and the levels of proteins were quantified. Results: Pregnancy swimming was able to prevent early metabolic changes induced by HI preserving the energetic balance, decreasing apoptotic cell death and astrogliosis as well as maintaining the levels of neurotrophins. At adult age, swimming preserved brain microstructure and improved the performance in the behavioral tests. Conclusion: Our study points out that swimming during gestation in rats could prevent prematurity related brain damage in progeny with high translational potential and possibly interesting cost-benefits. HIGHLIGHTS - Prematurity is a major cause of neurodevelopmental impairments;- Swimming during pregnancy reduces brain damage after HI injury;- Pregnancy is an important but underestimated preventive window.

Intra- and extra-axonal axial diffusivities in the white matter: Which one is faster?

A two-compartment model of diffusion in white matter, which accounts for intra- and extra-axonal spaces, is associated with two plausible mathematical scenarios: either the intra-axonal axial diffusivity Da,‖ is higher than the extra-axonal De,‖ (Branch 1), or the opposite, i.e. Da,‖ < De,‖ (Branch 2). This duality calls for an independent validation of compartment axial diffusivities, to determine which of the two cases holds. The aim of the present study was to use an intracerebroventricular injection of a gadolinium-based contrast agent to selectively reduce the extracellular water signal in the rat brain, and compare diffusion metrics in the genu of the corpus callosum before and after gadolinium infusion. The diffusion metrics considered were diffusion and kurtosis tensor metrics, as well as compartment-specific estimates of the WMTI-Watson two-compartment model. A strong decrease in genu T1 and T2 relaxation times post-Gd was observed (p < 0.001), as well as an increase of 48% in radial kurtosis (p < 0.05), which implies that the relative fraction of extracellular water signal was selectively decreased. This was further supported by a significant increase in intra-axonal water fraction as estimated from the two-compartment model, for both branches (p < 0.01 for Branch 1, p < 0.05 for Branch 2). However, pre-Gd estimates of axon dispersion in Branch 1 agreed better with literature than those of Branch 2. Furthermore, comparison of post-Gd changes in diffusivity and dispersion between data and simulations further supported Branch 1 as the biologically plausible solution, i.e. Da,‖ > De,‖. This result is fully consistent with other recent measurements of compartment axial diffusivities that used entirely different approaches, such as diffusion tensor encoding.

Apelin-13 treatment enhances the stability of atherosclerotic plaques.

BACKGROUND: Apelin is an endogenous peptidergic system which modulates cardiovascular function. Recent studies pointed out a fundamental contribution of apelin on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define a beneficial or deleterious role. To better understand apelin function on atherosclerosis, we aimed to investigate apelin-13 treatment effects on atherosclerotic plaques composition. DESIGN: Apolipoprotein E gene-deleted mice were fed on Western-type diet for 11 weeks. Atherosclerotic plaque formation was induced in the carotid artery by a shear stress modifier device, which exposes the same vessel to distinct patterns of shear stress enabling the formation of plaques with different composition. Mice were treated with apelin-13 (2 mg kg-1 day-1 ) or vehicle for the last 3 weeks. RESULTS: Apelin-13 treatment did not alter the lipid content of low shear stress- and oscillatory shear stress-induced plaques in the carotid. However, apelin-13 greatly ameliorated plaque stability by increasing intraplaque collagen content and reducing MMP-9 expression. Furthermore, apelin-13 decreased the infiltration of inflammatory cells (neutrophil and macrophage) and intraplaque reactive oxygen species content. Interestingly, apelin-13 treatment reduced total cholesterol, LDL levels and free fatty acid serum levels, while HDL, triglycerides serum levels were not significantly changed. CONCLUSIONS: Apelin-13 treatment for 3 weeks did not alter the lesion size, but it significantly enhanced the stable phenotype of atherosclerotic plaques and improved serum lipid profile. These results indicate that activation of apelin system decreases plaque vulnerability.

Alamandine abrogates neutrophil degranulation in atherosclerotic mice.

BACKGROUND: Neutrophil-mediated inflammation was recently identified as an active contributor to athero-progression. Therapeutic strategies inhibiting neutrophil degranulation or recruitment were hypothesized to positively impact on plaque vulnerability. In this study, we investigated whether treatment with the recently discovered agonist of the Mas-related G-coupled receptor type D (MrgD) alamandine would impact on neutrophil degranulation in vivo and in vitro. MATERIALS AND METHODS: Fifteen-week-old ApoE-/- mice were fed with a Western-type diet for an additional 11 weeks. After the first 2 weeks of diet, mice were surgically implanted with a carotid 'cast' device that alters the blood shear stress and induces different carotid plaque phenotypes. During the last 4 weeks before euthanasia, mice were randomly assigned to subcutaneously receive vehicle (NaCl 0·15 M) or alamandine (24 µg/kg/h) by micropump. For in vitro experiments, neutrophils were obtained after thioglycollate intraperitoneal injection in ApoE-/- mice. RESULTS: Treatment with alamandine was well-tolerated, but failed to affect lipid, macrophage, neutrophil or collagen content within carotid and aortic root plaques. Also, treatment with alamandine did not affect Th-cell polarization in lymphoid organs. Conversely, alamandine administration was associated with a reduction in serum levels of neutrophil granule enzymes, such as MMP-9 and MPO as well as MMP-9 content within aortic root plaques. In vitro, preincubation with alamandine dose-dependently abrogated PMA-induced neutrophil degranulation of MMP-9 and MPO. CONCLUSION: These results suggest that treatment with the MrgD agonist alamandine led to a reduced release of neutrophil granule products, potentially interfering with pro-atherosclerotic neutrophil activation.

Update on the role of angiotensin in the pathophysiology of coronary atherothrombosis.

BACKGROUND: Coronary atherothrombosis due to atherosclerotic plaque rupture or erosion is frequently associated with acute coronary syndromes (ACS). Significant efforts have been made to elucidate the pathophysiological mechanisms underlying acute coronary events. MATERIALS AND METHODS: This narrative review is based on the material searched for and obtained via PubMed up to August 2014. The search terms we used were as follows: 'angiotensin, acute coronary syndromes, acute myocardial infarction' in combination with 'atherosclerosis, vulnerability, clinical trial, ACE inhibitors, inflammation'. RESULTS: Among several regulatory components, the renin-angiotensin system (RAS) was shown as a key pathway modulating coronary atherosclerotic plaque vulnerability. Indeed, these molecules are involved in all stages of atherogenesis. Classically, the RAS is composed by a series of enzymatic reactions leading to the angiotensin (Ang) II generation and activity. However, the knowledge of RAS has expanded and become more complex. The discovery of novel components and their functions has revealed additional pathways that contribute to or counterbalance the actions of Ang II. In this review, we discussed on recent findings concerning the role of different angiotensin peptides in the pathophysiology of ACS and coronary atherothrombosis, exploring the link between these molecules and atherosclerotic plaque vulnerability. CONCLUSIONS: Treatments selectively targeting angiotensins (including Mas and AT2 agonists, ACE2 recombinant, or Ang-(1-7) and almandine in oral formulations) have been tested in animal studies or in small human subgroups, expanding the perspective in the ACS prevention. These novel strategies, especially in the counter-regulatory axis ACE2/Ang-(1-7)/Mas, might be promising to reduce plaque vulnerability and inflammation.

Gastric bypass in morbid obese patients is associated with reduction in adipose tissue inflammation via N-oleoylethanolamide (OEA)-mediated pathways.

Paradoxically, morbid obesity was suggested to protect from cardiovascular co-morbidities as compared to overweight/obese patients. We hypothesise that this paradox could be inferred to modulation of the "endocannabinoid" system on systemic and subcutaneous adipose tissue (SAT) inflammation. We designed a translational project including clinical and in vitro studies at Geneva University Hospital. Morbid obese subjects (n=11) were submitted to gastric bypass surgery (GBS) and followed up for one year (post-GBS). Insulin resistance and circulating and SAT levels of endocannabinoids, adipocytokines and CC chemokines were assessed pre- and post-GBS and compared to a control group of normal and overweight subjects (CTL) (n=20). In vitro cultures with 3T3-L1 adipocytes were used to validate findings from clinical results. Morbid obese subjects had baseline lower insulin sensitivity and higher hs-CRP, leptin, CCL5 and anandamide (AEA) levels as compared to CTL. GBS induced a massive weight and fat mass loss, improved insulin sensitivity and lipid profile, decreased C-reactive protein, leptin, and CCL2 levels. In SAT, increased expression of resistin, CCL2, CCL5 and tumour necrosis factor and reduced MGLL were shown in morbid obese patients pre-GBS when compared to CTL. GBS increased all endocannabinoids and reduced adipocytokines and CC chemokines. In morbid obese SAT, inverse correlations independent of body mass index were shown between palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) levels and inflammatory molecules. In vitro, OEA inhibited CCL2 secretion from adipocytes via ERK1/2 activation. In conclusion, GBS was associated with relevant clinical, metabolic and inflammatory improvements, increasing endocannabinoid levels in SAT. OEA directly reduced CCL2 secretion via ERK1/2 activation in adipocytes.

Treatment with recombinant tissue plasminogen activator (r-TPA) induces neutrophil degranulation in vitro via defined pathways.

Thrombolysis is recommended for reperfusion following acute ischemic stroke (AIS), but its effects on stroke-associated injury remain to be clarified. Here, we investigated the effects of recombinant tissue plasminogen activator (r-tPA) on neutrophil pathophysiology in vitro and in a case-control study with AIS patients submitted (n=60) or not (n=30) to thrombolysis. Patients underwent radiological and clinical examination as well as blood sampling at admission and after 1, 7 and 90days. In vitro, 30-min incubation with 0.1-1 mg/ml r-tPA induced neutrophil degranulation in different substrate cultures. Pre-incubation with kinase inhibitors and Western blot documented that degranulation was associated with activation of PI3K/Akt and ERK1/2 pathways in Teflon dishes and PI3K/Akt in polystyrene. In thrombolysed patients, a peak of neutrophil degranulation products (matrix metalloproteinase [MMP]-9, MMP-8, neutrophil elastase and myeloperoxidase), was shown during the first hours from drug administration. This was accompanied by serum augmentation of protective tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. An increased rate of haemorrhagic transformations on day 1 after AIS was shown in thrombolysed patients as compared to non-thrombolysed controls. In conclusion, r-tPA treatment was associated with in vitro neutrophil degranulation, indicating these cells as potential determinants in early haemorrhagic complications after thrombolysis in AIS patients.