Macrophages Promote Repair of Inner Hair Cell Ribbon Synapses following Noise-Induced Cochlear Synaptopathy.

Resident cochlear macrophages rapidly migrate into the inner hair cell synaptic region and directly contact the damaged synaptic connections after noise-induced synaptopathy. Eventually, such damaged synapses are spontaneously repaired, but the precise role of macrophages in synaptic degeneration and repair remains unknown. To address this, cochlear macrophages were eliminated using colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Sustained treatment with PLX5622 in CX3CR1 GFP/+ mice of both sexes led to robust elimination of resident macrophages (∼94%) without significant adverse effects on peripheral leukocytes, cochlear function, and structure. At 1 day (d) post noise exposure of 93 or 90 dB SPL for 2 hours, the degree of hearing loss and synapse loss were comparable in the presence and absence of macrophages. At 30 d after exposure, damaged synapses appeared repaired in the presence of macrophages. However, in the absence of macrophages, such synaptic repair was significantly reduced. Remarkably, on cessation of PLX5622 treatment, macrophages repopulated the cochlea, leading to enhanced synaptic repair. Elevated auditory brainstem response thresholds and reduced auditory brainstem response Peak 1 amplitudes showed limited recovery in the absence of macrophages but recovered similarly with resident and repopulated macrophages. Cochlear neuron loss was augmented in the absence of macrophages but showed preservation with resident and repopulated macrophages after noise exposure. While the central auditory effects of PLX5622 treatment and microglia depletion remain to be investigated, these data demonstrate that macrophages do not affect synaptic degeneration but are necessary and sufficient to restore cochlear synapses and function after noise-induced synaptopathy.SIGNIFICANCE STATEMENT The synaptic connections between cochlear inner hair cells and spiral ganglion neurons can be lost because of noise over exposure or biological aging. This loss may represent the most common causes of sensorineural hearing loss also known as hidden hearing loss. Synaptic loss results in degradation of auditory information, leading to difficulty in listening in noisy environments and other auditory perceptual disorders. We demonstrate that resident macrophages of the cochlea are necessary and sufficient to restore synapses and function following synaptopathic noise exposure. Our work reveals a novel role for innate-immune cells, such as macrophages in synaptic repair, that could be harnessed to regenerate lost ribbon synapses in noise- or age-linked cochlear synaptopathy, hidden hearing loss, and associated perceptual anomalies.

Pomegranate Peel Extract Decreases Plaque Necrosis and Advanced Atherosclerosis Progression in Apoe -/- Mice.

Atherosclerosis is a chronic lipid-driven inflammatory condition of the arteries and is a leading cause of stroke, myocardial infarction, and other peripheral arterial diseases. Plant products rich in polyphenols such as pomegranate juice and peel extract are known to have beneficial effects in suppressing atherogenesis. However, the mechanism of action and its effect on advanced atherosclerosis progression which results in adverse clinical outcomes are not well understood. Herein, we use a standardized hydroethanolic extract of Punica granatum (pomegranate) peel in the Apoe -/- a murine model of advanced atherosclerosis. It was observed that the pomegranate peel extract fed mice have decreased plaque necrosis and elevated lesional collagen content which was associated with a favorable metabolic profile including lowering of blood glucose, cholesterol, and triglyceride. The decrease in plaque necrosis was linked with increased lesional macrophage efferocytosis efficiency which was associated with enhanced expression of the efferocytosis receptor Mertk. Using in vitro studies, we show that pomegranate peel extract blocks the shedding of Mertk and preserves macrophage efferocytosis efficiency. These data identify a novel mechanism by which pomegranate peel extract promotes the resolution of inflammation in atherosclerosis.

Neutrophil DREAM promotes neutrophil recruitment in vascular inflammation.

The interaction between neutrophils and endothelial cells is critical for the pathogenesis of vascular inflammation. However, the regulation of neutrophil adhesive function remains not fully understood. Intravital microscopy demonstrates that neutrophil DREAM promotes neutrophil recruitment to sites of inflammation induced by TNF-α but not MIP-2 or fMLP. We observe that neutrophil DREAM represses expression of A20, a negative regulator of NF-κB activity, and enhances expression of pro-inflammatory molecules and phosphorylation of IκB kinase (IKK) after TNF-α stimulation. Studies using genetic and pharmacologic approaches reveal that DREAM deficiency and IKKß inhibition significantly diminish the ligand-binding activity of ß2 integrins in TNF-α-stimulated neutrophils or neutrophil-like HL-60 cells. Neutrophil DREAM promotes degranulation through IKKß-mediated SNAP-23 phosphorylation. Using sickle cell disease mice lacking DREAM, we show that hematopoietic DREAM promotes vaso-occlusive events in microvessels following TNF-α challenge. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases.

Hypercholesterolemia Impairs Clearance of Neutrophil Extracellular Traps and Promotes Inflammation and Atherosclerotic Plaque Progression.

Objective: Hypercholesterolemia-induced NETosis and accumulation of neutrophil extracellular traps (NETs) in the atherosclerotic lesion exacerbates inflammation and is causally implicated in plaque progression. We investigated whether hypercholesterolemia additionally impairs the clearance of NETs mediated by endonucleases such as DNase1 and DNase1L3 and its implication in advanced atherosclerotic plaque progression. Approach and Results: Using a mouse model, we demonstrate that an experimental increase in the systemic level of NETs leads to a rapid increase in serum DNase activity, which is critical for the prompt clearance of NETs and achieving inflammation resolution. Importantly, hypercholesterolemic mice demonstrate an impairment in this critical NET-induced DNase response with consequent delay in the clearance of NETs and defective inflammation resolution. Administration of tauroursodeoxycholic acid, a chemical chaperone that relieves endoplasmic reticulum stress, rescued the hypercholesterolemia-induced impairment in the NET-induced DNase response suggesting a causal role for endoplasmic reticulum stress in this phenomenon. Correction of the defective DNase response with exogenous supplementation of DNase1 in Apoe-/- mice with advanced atherosclerosis resulted in a decrease in plaque NET content and significant plaque remodeling with decreased area of plaque necrosis and increased collagen content. From a translational standpoint, we demonstrate that humans with hypercholesterolemia have elevated systemic extracellular DNA levels and decreased plasma DNase activity. Conclusions: These data suggest that hypercholesterolemia impairs the NET-induced DNase response resulting in defective clearance and accumulation of NETs in the atherosclerotic plaque. Therefore, strategies aimed at rescuing this defect could be of potential therapeutic benefit in promoting inflammation resolution and atherosclerotic plaque stabilization.

ERO1-PDI Redox Signaling in Health and Disease.

Significance: Protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductase 1 (ERO1) are crucial for oxidative protein folding in the endoplasmic reticulum (ER). These enzymes are frequently overexpressed and secreted, and they contribute to the pathology of neurodegenerative, cardiovascular, and metabolic diseases. Recent Advances: Tissue-specific knockout mouse models and pharmacologic inhibitors have been developed to advance our understanding of the cell-specific functions of PDI and ERO1. In addition to their roles in protecting cells from the unfolded protein response and oxidative stress, recent studies have revealed that PDI and ERO1 also function outside of the cells. Critical Issues: Despite the well-known contributions of PDI and ERO1 to specific disease pathology, the detailed molecular and cellular mechanisms underlying these activities remain to be elucidated. Further, although PDI and ERO1 inhibitors have been identified, the results from previous studies require careful evaluation, as many of these agents are not selective and may have significant cytotoxicity. Future Directions: The functions of PDI and ERO1 in the ER have been extensively studied. Additional studies will be required to define their functions outside the ER.

Iron Oxide Nanoparticles Affects Behaviour and Monoamine Levels in Mice.

Iron oxide (Fe2O3) nanoparticles (NPs) attract the attention of clinicians for its unique magnetic and paramagnetic properties, which are exclusively used in neurodiagnostics and therapeutics among the other biomedical applications. Despite numerous research findings has already proved neurotoxicity of Fe2O3-NPs, factors affecting neurobehaviour has not been elucidated. In this study, mice were exposed to Fe2O3-NPs (25 and 50 mg/kg body weight) by oral intubation daily for 30 days. It was observed that Fe2O3-NPs remarkably impair motor coordination and memory. In the treated brain regions, mitochondrial damage, depleted energy level and decreased ATPase (Mg2+, Ca2+ and Na+/K+) activities were observed. Disturbed ion homeostasis and axonal demyelination in the treated brain regions contributes to poor motor coordination. Increased intracellular calcium ([Ca2+]i) and decreased expression of growth associated protein 43 (GAP43) impairs vesicular exocytosis could result in insufficient signal between neurons. In addition, levels of dopamine (DA), norepinephrine (NE) and epinephrine (EP) were found to be altered in the subjected brain regions in correspondence to the expression of monoamine oxidases (MAO). Along with all these factors, over expression of glial fibrillary acidic protein (GFAP) confirms the neuronal damage, suggesting the evidences for behavioural changes.

Iron Oxide Nanoparticles Induces Cell Cycle-Dependent Neuronal Apoptosis in Mice.

Iron oxide (Fe2O3) nanoparticles (NPs) with its unique magnetic and paramagnetic properties are popular in biomedical applications. Some of their neurotoxic mechanisms due to repeated administration are proven. However, we speculate that the neuronal damage might be due to apoptosis resulting from unusual cell cycle entry. Moreover, iron accumulation has been shown to be closely associated with most of the neurodegenerative disorders. Thus, in the current study, mice were orally (po) treated with the Fe2O3-NPs to investigate cell cycle-associated events/components and occurrence of apoptosis. A subsequent increase in oxidant levels was observed with the iron accumulation due to Fe2O3-NPs exposure. The accumulated ß-amyloid and reduced level of cdk5 seem to aid in the cell cycle entry and forcing progression towards apoptosis. Expression of Cyclin D1 and pRb (Ser 795) indicate the cell cycle re-entry of neurons. Overexpression of RNA Pol II and PARP cleavage suggests DNA damage due to Fe2O3-NPs exposure. Further, hyperphosphorylation of p38 (Thr 180/Tyr 182) confirms the activation of DNA damage-dependent checkpoint. Expression patterns of pro- and anti-apoptotic markers, TUNEL and TEM indicate the occurrences of apoptosis.

Recurrent exposure to ferric oxide nanoparticles alters myocardial oxidative stress, apoptosis and necrotic markers in male mice.

The cardiotoxicity of iron oxide nanoparticles (Fe2O3-NPs) in mice was investigated. The mice were intraperitoneally administered with Fe2O3-NPs at the dose of 25 and 50 mg/kg bw for 30 days at seven days interval. In vivo MRI analysis reveals the Fe2O3-NPs accumulation in the cardiac system. Also, serum iron estimation and Prussian blue staining confirms the iron deposition in circulatory system. Cardiac dysfunction was assessed by ECG analysis and further validated by evaluating the functional markers such as cardiac Troponin-1 (cTnI) expression, AChE activity and levels of LDH and CK-MB in cardiac tissue. Fe2O3-NPs exposure disturbs the balance between the oxidants and antioxidants resulting in oxidative myocardial damages. In consequence, damaged mitochondria, diminished ATP level and NOX4 over expression were observed in the intoxicated groups indicating the role of Fe2O3-NPs in oxidative stress. A dose dependant increase in oxidative stress mediates apoptosis through upregulation of Bax, cytochrome c and cleaved caspase 3 in the 25 mg/kg treated group. Sustained oxidative stress suggest the occurrence of necrosis in addition to apoptosis in 50 mg/kg treated group evidenced by altered expression pattern of cleaved PARP, cytochrome c, Bax and cleaved caspase 3. In addition, triphenyl tetrazolium chloride (TTC) staining confirms cardiac necrosis in 50 mg/kg Fe2O3-NPs treated group.

Neurobehavioural Toxicity of Iron Oxide Nanoparticles in Mice.

Iron oxide nanoparticles (Fe2O3-NPs) are widely used in various biomedical applications, extremely in neurotheranostics. Simultaneously, Fe2O3-NP usage is of alarming concern, as its exposure to living systems causes deleterious effects due to its redox potential. However, study on the neurobehavioural impacts of Fe2O3-NPs is very limited. In this regard, adult male mice were intraperitoneally administered with Fe2O3-NPs (25 and 50 mg/kg body weight) once a week for 4 weeks. A significant change in locomotor behaviour and spatial memory was observed in Fe2O3-NP-treated animals. Damages to blood-brain barrier permeability by Fe2O3-NPs and their accumulation in brain regions were evidenced by Evan's blue staining, iron estimation and Prussian blue staining. Elevated nitric oxide, acetylcholinesterase, lactate dehydrogenase leakage and demyelination were observed in the Fe2O3-NP-exposed brain tissues. Imbalanced levels of ROS generation and antioxidant defence mechanism (superoxide dismutase and catalase) cause damages to lipids, proteins and DNA. PARP and cleaved caspase 3 expression levels were found to be increased in the Fe2O3-NP-exposed brain regions which confirms DNA damage and apoptosis. Thus, repeated Fe2O3-NP exposure causes neurobehavioural impairments by nanoparticle accumulation, oxidative stress and apoptosis in the mouse brain.

Repeated exposure to iron oxide nanoparticles causes testicular toxicity in mice.

The aim of this study was to determine whether repeated exposure to iron oxide nanoparticles (Fe2 O3 -NPs) could be toxic to mice testis. Fe2 O3 -NPs (25 and 50 mg/kg) were intraperitoneally administered into mice once a week for 4 weeks. Our study showed that Fe2 O3 -NPs have the ability to cross the blood-testis barrier to get into the testis. The findings showed that exposure resulted in the accumulation of Fe2 O3 -NPs which was evidenced from the iron content and accumulation in the testis. Furthermore, 25 and 50 mg/kg Fe2 O3 -NPs administration increased the reactive oxygen species, lipid peroxidation, protein carbonyl content, glutathione peroxidase activity, and nitric oxide levels with a concomitant decrease in the levels of antioxidants-superoxide dismutase, catalase, glutathione, and vitamin C. Increased expression of Bax, cleaved-caspase-3, and cleaved-PARP confirms apoptosis. Serum testosterone levels increased with increased concentration of Fe2 O3 -NPs exposure. In addition, the histopathological lesions like vacuolization, detachment, and sloughing of germ cells were also observed in response to Fe2 O3 -NPs treatment. The data from our study entailed that testicular toxicity caused by Fe2 O3 -NPs exposure may be associated with Fe2 O3 -NPs accumulation leading to oxidative stress and apoptosis. Therefore, precautions should be taken in the safe use of Fe2 O3 -NPs to avoid complications in the fertility of males. Further research will unravel the possible molecular mechanisms on testicular toxicity of Fe2 O3 -NPs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 594-608, 2017.