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Cell Culturing on 3D Printed LCEs
The purpose of this study is to culture cells on 3D printed structures known as liquid crystal elastomers (LCEs) using templates from microscope scanned tissue specimens. LCEs are composed of a long crosslink of polymers that have a porous property to them that will aid in growth and proliferation of the glial cells once they are seeded into the material. The process by which this is possible is photo curing resin of different types by using multiple formulations of elastomers that contain varying polymerizers. Two main forms of printing are used to accomplish this; an SLA UV curing printer and the use of a custom designed microscope setup. The SLA UV curing printer cures on a layer-by-layer basis at ~30 micron resolution. While the custom designed setup allows for the use of a laser scanning microscope which is capable of providing finer resolution for the LCEs. So far, through the use of the SLA UV curing printer, our lab has been able to print structures of tissue that contain all the characteristics that would make the growth of glial cells a feasible task. The structures were printed using modified materials which contain photo-initiators that enable the material to undergo physical property changes when they react with the UV light from the printer. More specifically, it was a compound by the name of 6arms-alpha (modified), which contains 10% of liquid crystal and has a high viscosity. The importance of this research is to provide a way to study neurons in arrangements that emulate real tissue, which could have a significant impact in the future of bioimplants.
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Impact of the Methyl Donor Betaine on Repairing Mitochondria in Neurodegenerative Disease
In Alzheimer’s disease (AD) and multiple sclerosis (MS) histone methylation is severely reduced. This hypomethylation leads to aberrant gene expression that brings about disease-causing plaque and lesion buildup within the central nervous system. It is known that this aberrant gene expression affects mitochondria in these diseases. I hypothesized that introducing methyl donors could prevent hypomethylation of histone H3, as well as dysfunction of mitochondria in mouse models of AD and MS. I tested the effects of the methyl donor betaine (also known as trimethylglycine). We administered betaine in drinking water to the APP/PS1 mouse model of AD, and the Cuprizone mouse model of MS for four weeks. The aim of this study was to measure the protective effects of the methyl donor betaine on mitochondrial complexes downregulated in neurodegenerative diseases. Protein was isolated from the brains of mice with and without betaine. The levels of different mitochondrial complex genes in the presence and absence of betaine were measured using Western blotting, JC1 staining, immunohistochemistry (IHC), and densitometry. This research has important implications for developing new therapies to treat AD and MS.
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Actigraphy-Based Circadian Measures and Cerebrospinal Fluid Biomarkers of Neurodegeneration in Alzheimer’s Disease with Mild Cognitive Impairment
Although recent data implicates sleep and circadian disruption to neurodegeneration in Alzheimer’s Disease (AD), the association of objective circadian biomarkers and neurodegeneration remains understudied. We hypothesize that actigraphy-based circadian measures are associated with cerebrospinal fluid (CSF) biomarkers of neurodegeneration in those mild cognitive impairment due to AD (MCI-AD).Eighteen patients with CSF biomarker-confirmed MCI-AD underwent actigraphy monitoring generating the following circadian measures: amplitude, F-ratio and mesor and morning collection of CSF biomarkers of neurodegeneration (Aβ42,t-tau,p-tau). Linear models were used to evaluate the association of circadian and CSF measures; logarithmic transformations were performed on neurodegenerative markers for greater normality. Analysis was performed using SAS software. A significance level of 0.05 was assumed for all tests. Eighteen MCI-AD patients who were 68± 6.2 years, 44% female, with median AHI=12 and underwent actigraphy monitoring for 8.2+/-3.2 days were included. There was no significant association of circadian measures and Aβ42 nor with mesor and neurodegeneration biomarkers. Amplitude was associated with both p-tau and t-tau, such that each 10 unit increase in amplitude resulted in a predicted increase in p-tau of 8% (95% CI: 1%-15%, p=0.018) and an increase of 13% (3%-23%; p=0.01) in t-tau. F-ratio was positively associated with p-tau and t-tau; each 1000 unit increase in F-ratio resulted in a predicted 12% (4%-22%; p=0.007) increase in P-tau and 20% (6%-35%; p=0.005) increase in t-tau. Associations of these circadian measures and CSF levels of p-tau and t-tau remained statistically significant after adjustment for age and sex. Among patients with symptomatic MCI stages of AD, objective measures of circadian rhythm disruption are associated with CSF-based biomarkers of neurodegeneration even after consideration of age and sex.
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Treating Neuropathology with the Novel Hormone Irisin
Alzheimer’s disease is characterized by neuroinflammation and neural accumulation of pathological tau. One candidate for treatment is a recently discovered hormone irisin, which is a cleaved product from the fibronectin type III domain-containing protein 5 (FNDC5) transmembrane protein. We have preliminary data showing that weekly injections of recombinant irisin decreased neuroinflammation and tau pathology in 4-month old female htau mice that express a mutant, humanized gene associated with late-onset Alzheimer’s disease. In the current study, we determined how changes in treatment frequency and age affected neuropathology, inflammation, and FNDC5 levels in htau mice and their wildtype littermates that lack any form of tau.
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Impact of Physical Activity on CNS Mitochondrial Metabolism School of Biomedical Sciences, Kent State University Dept. of Biological Sciences, Kent State University Brain Health Research Institute, Kent State University
Multiple sclerosis (MS) is a complex neurodegenerative disease of the central nervous system (CNS) and dysfunctional mitochondria in neurons have been suggested to play a role. Hemoglobin is present in cortical neurons and levels are altered in MS cortex. The role of hemoglobin is unclear, however, hemoglobin β (Hbb) subunit levels are increased in mitochondrial fraction of MS cortex suggesting a potential role of hemoglobin in mitochondrial respiration. In fact, Hbb is tightly bound to chromatin and mediates a signaling mechanism that supports neuronal energetics involving the trimethylation of histone H3 on lysine 4 (H3K4me3), a histone mark that activates transcription of mitochondrial genes and increases mitochondrial respiratory capacity. We are excited by the potential influence that exercise may have on Hbb expression in the CNS and its impact on neuronal energetics. Thus, we compared exercise and sedentary lifestyles on neuronal Hbb expression and mitochondrial metabolism using Sprague Dawley rats. Rats aging 6-8 weeks-old were provided running wheels for a 7 week period. The Hbb expression was shown to be increased in exercised rats compared to sedentary rats. We also observed that increased Hbb expression correlated with an increase in basal neuronal mitochondrial respiration and increased mitochondrial respiration was linked to elevated levels of the neuronal mitochondrial metabolite N-acetylaspartate (NAA). Understanding regulatory mechanisms that impact Hbb expression and how Hbb supports neuronal mitochondria could lead to new therapeutic approaches (including exercise) to treat neurodegenerative diseases including MS, Alzheimer’s, and Parkinson’s where dysfunctional mitochondria contribute to disease activity.
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Ferret Odor Induces Novel Gene Pathways in the Ventromedial Hypothalamus
Nearly 40% of Americans are obese and this can be countered by increasing calories burned. Muscle thermogenesis is a possible untapped source for increasing energy expenditure. Elevating thermogenesis increases energy expenditure and is linked to weight loss. Our research program has shown that exposing rats to predator odor (PO) induces a rapid and robust increase in muscle thermogenesis that peaks by 30 min and settles to near baseline after 4 hours. We hypothesize that this is mediated in the brain by the ventromedial hypothalamus (VMH) because of the importance of the VMH in regulating body weight, muscle metabolism, sympathetic nervous system activity, and the behavioral response to predator threat. We investigated possible PO-induced changes in the VMH by exposing rats to predator odor for 30 minutes to 4 hours. Tissue samples were taken from the VMH, and mRNA expression was measured using qPCR, showing an increase in Sirt 1 and Bdnf, but not SF1, after 4 hours of PO exposure. RNA-sequencing analyses identified 164 differentially expressed genes (DEGs), 65 enriched gene ontology terms, and 28 genome pathways revealing themes of immune response, oxidative stress, and synaptic plasticity. This allowed us to conclude that PO exposure causes upregulation of novel genes pathways.
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Effects of Early Life Neglect on Adolescent Cocaine Use and Expression of Fibroblast Growth Factor-2 in the Brain
Early-life adversity (ELA) increases the risk for development of psychopathologies in adolescence and adulthood. Childhood neglect is a potent form of ELA and can be modeled through neonatal maternal separation. Maternal separation has been shown to alter cognition, learning and memory later in life. Here, we examined the contribution of maternal separation to the development of substance use disorder in adolescence, and whether changes in regional protein expression in the brain persist into adulthood. First, male and female rats exposed to maternal separation were compared to controls for changes in localized expression of the developmental growth factor, fibroblast growth factor-2 (FGF2), in several brain regions using immunohistochemistry. FGF2 is known to be upregulated by cocaine experience, is protective against fear over-expression and is a candidate biomarker for vulnerability and resilience to development of comorbid psychopathologies. Next, we examined whether maternal separation impacted sensitivity in adolescence to cocaine using the conditioned place preference paradigm, in which one of two chambers is associated with cocaine. Finally, we determined whether maternal separation altered regional FGF2 expression in the brain during adulthood, and if cocaine experience in adolescence had an impact. We are currently processing the brain tissue to examine regional FGF2 expression and have completed the conditioned place preference protocol. Our results will determine whether FGF2 is a potential risk or resilience factor for the development of psychopathologies and will pave the way for future studies examining comorbid addiction and fear disorders.
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Mitochondrial Dynamics in Neurodegenerative Mouse Models
Neurodegenerative diseases are becoming more common in today’s world. Two of the more prominent diseases that fall into this category are multiple sclerosis (MS) and Alzheimer’s disease (AD). A hallmark for both of these diseases is mitochondrial dysfunction. Mitochondrial dysfunction causes an increase in the number of damaged mitochondria, which is linked to a decrease in the energy supply for the cell. Mitophagy is a mechanism known for eliminating damaged mitochondria and is impaired in neurodegenerative diseases. This causes an accumulation of damaged mitochondria in the cell leading to loss of function. We have also shown that a dietary factor known as betaine, is able to restore energy production within the dysfunctional mitochondria. In the present study, we are investigating the role of betaine in both AD and cuprizone mouse models, hypothesizing that betaine will restore energy production by eliminating damaged mitochondria using mitophagy.
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The Role of the Locus Coeruleus on Regulating Sickness-like BehaviorsElevated peripheral cytokines result in sickness and depression-like behaviors by signaling through the vagus nerve. Stimulation of the vagus nerve activates noradrenergic neurons in the nucleus of the solitary tract (NTS) and release of norepinephrine (NE) at projection sites that regulate behavior. Previous studies indicate that neurons in the locus coeruleus (LC) inhibit neurons in the NTS. This led to the hypothesis that LC neurons may act as a brake to inhibit sickness behaviors. To test this hypothesis, rats underwent baseline behavioral testing before being injected with either saline or DSP4, a neurotoxin that lesions LC neurons. Three weeks later animals underwent further behavioral testing before all animals received live E.coli injections and sickness behaviors were recorded. We expected lesioning the LC would result in greater sickness behaviors following E.coli by removing the inhibitory regulation of NTS projection neurons; however, lesioned animals had similar body weights, food intake, and sucrose intake compared to controls following E.coli challenge. Interestingly, DSP4 treated rats showed greater anxiety-like behaviors in an open field test three weeks following DPS4 and less sickness behaviors in an open field following E.coli compared to controls. The results did not support our initial hypothesis, but do support a role for LC neurons in regulating sickness and/or depression/anxiety-like behaviors. Future studies will look more into the role the LC plays in regulating behaviors and the interactions between NTS, LC, and prefrontal cortex (an area important for emotional and motivated behaviors). |
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Bryan Soth Abstracts for Research Symposium - RACK1 Rescue Project
Down syndrome occurs due to triplication of human chromosome 21, and results in many phenotypic defects including slow wound healing, intellectual disability and reduced muscle tone. Here we focus on the role of adhesions in this disorder, which are involved in tissue development, cell migration, and memory formation. Previous work from our lab demonstrates that fibroblasts from individuals with Down syndrome have increased focal adhesions, which are multi-protein complexes that link the extracellular matrix to the intracellular cytoskeleton. We also found that the area, perimeter and motility of Down syndrome fibroblasts is altered. Taken together, these data suggest that increased adhesion may lead to reduced cellular motility in Down syndrome. Here we show that RACK1, a scaffolding protein and member of the focal adhesion complex, is overexpressed in Down syndrome fibroblasts. We also demonstrate that RACK1 can be knocked down with shRNA in Down syndrome fibroblasts to levels comparable to apparently healthy control cells. We are currently investigating whether knocking down RACK1 to control values is able to rescue the phenotypic defects present in Down syndrome fibroblasts, such as cell size, cell shape, and motility. Furthermore, substrate type is known to have a profound effect on protein expression and differentiation, and we have preliminary data that the substrate affects the expression level of RACK1 differentially in Down syndrome fibroblasts. Examining the role of RACK1 in Down syndrome is relevant for understanding the phenotypic defects of individuals with this disorder and may also provide insight into therapeutic and clinical treatments.
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Identification of Opioid Receptors in Mixed Cortical Cultures
Introduction: The six layers of the rat cerebral cortex consists of around twenty-one million cells which can be sub-divided into distinct neuronal and glial populations (e.g. GABAnergic, Glutaminergic, Astrocytic). These neuronal populations are differentially targeted by both licit and illicit drugs to alter cellular states, excitability, and/or behavioral outcome. Opioids alter cortical excitability through inhibition of GABAnergic inhibitory interneurons leading to disinhibition of glutaminergic neurons and downstream excitation of dopaminergic neurons. Resulting in altered reward evaluation and opioid induce analgesia. Objective: To use Immunocytochemistry - Immunofluorescence to identify the key cellular targets of opioids in the cortex. Methods: Cortical neurons were isolated from (P0) rat pups, cultured on glass coverslips and allowed to form mature synapses over 12 days. On days 13-20 cultures were Immuno-labeled with neuronal (Anti-NeuN), Astrocytic (Anti-GFAP), GABAnergic (Anti-GAD67) and Mu1 Opioid receptor (Anti-OPRM1) conjugated antibodies and imaged at 405nm, 488nm, 550nm, and 633nm respectively on an inverted confocal microscope. Results: Opioid receptors were found to be co-localized with GAD67 and NeuN labeling with little to no detectable expression on astrocytes or other neuronal subpopulations. Conclusion: These data support previous evidence that opioids selectively act on specific subpopulations of cortical neurons that suppress and limit cortical excitability.
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Methods to Create a Multi Modal Imaging Probe
Magnetic resonance imaging (MRI) scans use a combination of radio waves and magnetic fields to create an image of tissues within the body. While MRI is proven effective it only has limited use as to which tissue structures can be accurately resolved. Using contrast agents in MRI, it is possible to gain enhanced detail in acquired images to help resolve disease symptoms, tissue activity, and improve the signal of tissues during a scan. Contrast agents are used in patients with Multiple Sclerosis to detect lesions in brain tissue. Two issues with current contrast agents is their lack of specificity and toxicity at relatively low doses. Recently, a new a gadolinium-based nanoparticle (GdNP) has been developed. GdNP is approximately ten times stronger than typical contrast agents allowing it to provide the same contrast enhancement with ten percent of the typically required concentration. Targeting agents further decrease the number of molecules needed for desired image enhancement by binding to specific areas of the tissues being studied. This research is based around developing and evaluating nanoparticle-targeting agents as contrast enhancing probes with tissue specificity. The albumin binding protein, Evans blue (EB), have been attached to contrast enhancing nanoparticles. EB targets serum albumin (SA) in the blood vessels when injected intraperitoneally. We present in vivo MRI and microscopic data detailing development and usage of this new probe designed to enhance signal from the neurovasculature of a mouse brain.
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The Effects of Human Chorionic Gonadotropin on the Onset and Severity of Kiantic-acid Induced Seizure Activity
Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein involved in reproductive function known to bind the luteinizing hormone receptor (LHR). LHR activation has been shown to inhibit function at high doses. Importantly, in the APP/PS1 Alzheimer’s disease mouse model, known to have increased susceptibility to seizures, we have observed that mortality due to seizures is significantly reduced in animals treated with intracerebral (ICV) hCG. Therefore, to more directly address if hCG has anti-convulsant effects we used a model of epilepsy (kiantic acid administration) to induce convulsions in the presence and absence of chronic ICV hCG treatment in WT and APP/PS1 mice. The seizure induction and severity were scored using a seizure scale and neuronal changes/damage was determined by Tunnel staining. Our preliminary data in APP/PS1 suggests hCG may reduce susceptibility and severity of seizures in this model of epilepsy.
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Evaluation of the Neuroprotective Properties of n-acetylaspartate in the Cuprizone Mouse Model of MS.
Multiple sclerosis (MS) is an autoimmune disorder characterized by the demyelination of neurons in the central nervous system. In MS, oligodendrocytes, the cells that wrap axons in a protective coating called myelin that allows for efficient cell signaling, are degraded. Due to high concentrations of n-acetylaspartate (NAA) found in healthy oligodendrocytes, it is thought to be utilized as a lipid source for myelin synthesis, and could prove to be a beneficial supplement for those who suffer from MS. In this study we examined what effect NAA had on myelin formation in the cuprizone mouse model of MS. We treated the mice with NAA and then determined the concentration of NAA in the brain via high-performance liquid chromatography (HPLC). Myelin staining, imaging, and densitometry were performed in order to determine the quality and density of myelin. We also looked at gene activity involved in mitochondrial biogenesis, as well as examining several behavioral phenotypes.
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The Effect of Inhibiting IL-1β Release on Fear Aquisition and Fear Memory
Chronic stress induces anxiety-like behaviors, including contextual fear memory. In the presence of stress, microglia, the immune cells of the brain, release the proinflammatory cytokine interleukin-1β (IL-1β). Previous studies have demonstrated that a slight increase in IL-1β can enhance fear memory in rats. However, an excess or lack of IL-1β can dampen fear memory. This study sought to examine the role of IL-1β on fear acquisition and fear memory under chronic stress conditions. To do so, the effect of the drug minocycline on this pathway was investigated. Minocycline has been previously demonstrated to inhibit microglial activation, thus halting release of IL-1β. This investigation hypothesized that minocycline administration in stressed rats would result in dampened fear memory. Rats were separated into stress and no-stress groups, then were either administered minocycline or saline 1h prior to fear conditioning. In conditioning, fear acquisition was measured and analyzed. The data suggest memory acquisition was intact. Additionally, there was a significant interaction of time and chronic stress in which chronic stress enhances fear acquisition. Fear memory was analyzed 24h post fear conditioning by measuring freezing behavior in the same context. Unexpectedly, results show no significant effect of chronic stress or a significant interaction of chronic stress and minocycline on fear memory. However, the data suggest minocycline significantly dampens contextual fear memory in rats. Previous studies have shown that increased IL-1β in the basolateral amygdala (BLA) results in dampened fear memory. Thus, future studies will further investigate effects of inhibiting IL-1β release in the BLA.
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