Pharmacology, Toxicology and Environmental Health Commons^™

The Aryl Hydrocarbon Receptor Regulates An Essential Transcriptional Element In The Immunoglobulin Heavy Chain Gene, Michael J. Wourms

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2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that inhibits immunoglobulin (Ig) expression and Ig heavy (IgH) chain gene transcription. Transcription of the IgH gene involves several regulatory elements including the 3'lgh regulatory region (3'lghRR) which is composed of four enhancers (hs3A, hs1,2, hs4, and hs3B). Dioxin responsive elements (DRE) in the hs4 and hs1,2 enhancers of the 3lghRR that bind the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates dioxin sensitive genes suggest that the 3'lghRR may be a transcriptional target of TCDD. The current study utilized an IgA secreting mouse …

Role Of Na+K+2cl- Co-Transporters In Insulin Secretion, Saeed Alshahrani

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The objective of this study is to investigate the role of intracellular chloride concentration ([Cl^-]i) in insulin secretion in vivo and in vitro.

The maintenance of high [Cl^-]i in insulin-secreting pancreatic β-cells plays an important role in glucose-induced depolarization and insulin secretion. Insulinotropic glucose concentrations i.e., >6 mM induces electrical activity in β-cells which results in insulin secretion. This electrical activity is mainly due to electrogenic efflux of Cl^- from β-cells through volume-regulated anion channels (VRAC). The solute carrier family 12 group A member 1 (Slc12a1) and 2 (Slc12a2) encode several splice variants of the …

Elucidating Transcription Factor Regulation By Tcdd Within The Hs1,2 Enhancer, Sharon D. Ochs

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Immunoglobulin heavy chain (IgH) expression and Ig secretion is inhibited by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). Within the IgH gene, the 3' IgH regulatory region (3'IgH RR) has been identified as a transcriptional target of TCDD. The 3'IgH RR, which in part regulates transcription of the IgH gene, is composed of four enhancers in the mouse: hs3a; hs1,2; h3b; hs4 and three enhancers in the human: hs3a; hs1,2; hs4. In humans the hs1,2 enhancer has an invariant sequence (IS) containing a DRE, NF-κB, NF1 and AP-1 binding site. Also, the enhancer has an AP1.ETS and Oct …

Evaluation Of Cm-2,525 As A Neuroprotectant Against Sarin: A Comparison With 8-Oh-Dpat, Amanda R. Furman

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Exposure to the chemical warfare agent sarin produces long term neurological deficits. The long term medical consequences could be averted with the development of neuroprotectants to preserve brain function. In our mouse model, the combination of the carboxylesterase inhibitor 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP) with the organophosphorus (OP) nerve agent sarin was used to render mice more sensitive to poisoning and reduce the amount of hydrogen fluoride cleaved from sarin during binding to esterases. Since carboxylesterase acts as a scavenger, reducing the levels causes sarin to have a greater inhibition effect on acetylcholinesterase. These smaller doses permitted the use of doses similar to …

Use Of Phage Display Libraries To Select For B-Cell Receptor-Specific Peptides Of Chronic Lymphocytic Leukemia Cells, Richard M. Chou

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Peptides with high affinity to the B-cell receptor (BCR) fused to a toxin could be an effective therapy for Chronic Lymphocytic Leukemia (CLL) patients. We screened captured BCR of a CLL patient with peptides from 7 and 12-mer phage display libraries, using two strategies. Lymphocytes from two patients diagnosed with CLL expressing two different unmutated VH genes (VH 1-3 and VH4-34, respectively) were used. Membrane BCRs were obtained from patient CLL cells by lysis, identified by western blot, semi-quantified and screened with phage libraries. The first strategy involved using patient VH4-34 BCRs which were captured using goat anti-human IgM to …

Light-Limited Access To Fructose Alters Metabolic Function And Adipose Tissue Catecholaminergic Activity In Mice, Naima Salem Rodwan

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Consumption of high levels of fructose produces glucose intolerance, sympathetic nervous system activation, and renal dysfunction. A diet high in fructose may play a role in the worldwide epidemic of obesity and diabetes. The aim of this study was to test the hypothesis that time (light/dark) limited access to fructose influences body fat, metabolic function, and adipose tissue catecholaminergic activity in mice. Male C57BL/6 mice were given standard chow and assigned to one of three groups: Control (n=10, water 24h); FL (n=11, 10% fructose solution during 12h light period); and FD (n=10, 10% fructose solution during 12h dark period). Metabolic …

Enhanced Angiotensin Ii-Induced Cardiac And Aortic Remodeling In Ace2 Knockout Mice, Mahmoud Alghamri

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Activation of the renin angiotensin system (RAS) is a well-known driving force, governing the aggravation and progression of CVD and associated target organ damage. Angiotensin (Ang) converting enzyme 2 (ACE2), a new member within the RAS family, was first cloned from the ventricle of patient with heart failure. ACE2 was shown to be upregulated under pathological conditions. We hypothesize that loss of ACE2 negatively affects cardiac function and exacerbates hypertrophic cardiomyopathy and aortic remodeling in response to Ang II. Eight weeks old ACE2 knock out (KO) and wild type (WT) mice were infused with Ang II s.c. (1000 ng/kg/min for …

Response Of Monovalent Cation Transporters To Pro-Apoptotic Protein Kinase C Modulators In Human Lens Epithelial Cells, Michael Anthony Lepera

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Protein kinase inhibition by staurosporine causes apoptotic volume decrease involving potassium (K) channels in immortalized human B3 lens epithelial cells (LECs). Here, the effect of two pro-apoptotic protein kinase C (PKC) inhibitors [12-O-tetradecanoyl-phorbol-13-acetate (TPA) and chelerythrine (CET)] were studied on membrane K transport in a fetal human LEC line (FHL124) by western blotting, immunofluorescence, ion flux, ATP, apoptosis, and biotinylation assays. Long term TPA exposure (0-6 h) inhibited 75% of Na-K-2Cl cotransport (NKCC). In contrast, short term (0-20 min) exposure to 50 μM CET reduced Na/K pump and NKCC by >90% and >70%, respectively, without retrieval from the membrane into …

Differentiation Of Megakaryocytes/Platelets And Neurons From Human Endometrial Stromal Progenitor Cells, Jinju Wang

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Human endometrium is a high dynamic tissue that contains stem/progenitor cells. These endometrial stromal progenitor cells (hESCs) have been differentiated into a number of mesodermal lineages. There is limited information on differentiating hESCs into neurons, and no information on differentiating hESCs into megakaryocytes (MKs) and platelets (PLTs). The aim of this work was to investigate the possibility of differentiating hESCs into two distinct lineages: MKs, with subsequent PLT formation, and neurons.

We isolated hESCs from human endometrial tissue and cultured the cells for 4-6 passages. Before each differentiation experiment, the purity of hESCs was confirmed by flow cytometry analysis which …

Role Of Hyperglycemia And Aldosterone On Renal Ace2 And Albuminuria In Db/Db Mice, Harshita Chodavarapu

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Diabetic nephropathy (DN) is one of the microvascular complications of type II diabetes and a leading cause for the development of end stage renal disease. Renin angiotensin system (RAS) plays a pivotal role in the development and progression of diabetic nephropathy. Hyperglycemia activates RAS and increases tissue and circulating levels of angiotensin II (Ang II) and aldosterone. A new component of RAS, angiotensin converting enzyme 2 (ACE2), has been shown to be renoprotective in early stages of diabetes. The aim of this study is to test the hypothesis that strict glycemic control using rosiglitazone or treatment with mineralocorticoid receptor (MR) …

Analysis Of Archived Dried Blood Spots By Mass Spectrometry For Vitamin D And Real-Time Pcr For Its Enzymes And Receptor, Amod N. Joshi

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Vitamin D is an essential micronutrient required for maintenance of the skeletal system and its deficiency leads to diminished availability of calcium for maintaining bone mineralization. Vitamin D deficiency (VDD) during pregnancy and/or in early infancy can lead to rickets and a fragile bone state resulting in fractures with minimal forces. Such multiple unexplained fractures (MUF) present in infants at less than 6 months of age are often misdiagnosed as cases of child abuse. Therefore, it is important to evaluate the status of vitamin D in early infancy. The analysis of dried blood spots (DBS) provides a unique screening method …

Response Of Neuroinflammatory And Neurodegenerative Markers Following Sub-Lethal Sarin Exposure And Subsequent Treatment Via An In-Vivo Caspase Inhibitor, Douglas F. Chmura

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Organophosphorus nerve agents are amongst the most deadly chemical compounds ever synthesized. Sarin is an organophosphate (OP) ester that irreversibly forms a phosphoester bond at the active site of acetylcholinesterase and thereby induces a rapid and lethal cholinergic crisis. It remains an active threat to vulnerable civilian populations due to its ease of synthesis and known use by rogue nations and terrorist groups. Death is the most extreme consequence of sarin toxicity. Current treatments fail to provide protection against progressive cognitive impairments years after mild exposure. Q-VD-OPh is an in-vivo caspase inhibitor with potent anti-apoptotic and anti-inflammatory properties. In this …

Synthesis, Characterization And Manipulation Of Creighton Silver Nanoparticles For Future Cytotoxicity Studies, Sesha Lakshmi Paluri

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Nowadays, 24% of the nanomaterial-based consumer products contain silver nanoparticles (AgNPs) and exploit the well-known antimicrobial properties of silver. Although AgNPs have a wide range of biomedical research and industrial applications, very little is known about their toxicity. The main goal of this study is to synthesize, characterize and manipulate Creighton colloidal AgNPs for future cytotoxicity studies. These "naked" AgNPs were free from chemically aggressive capping/stabilizing agents, reaction byproducts or organic solvents. To achieve this goal, colloidal AgNPs were successfully: a) synthesized in large volumes (5L) using a slightly modified Creighton method by the reduction of silver nitrate with sodium …

Low Dose Nerve Agent Sarin Causes Dilated Cardiomyopathy And Autonomic Imbalance In Mice, Swapnil Vijay Shewale

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Sarin, a lethal chemical nerve agent, may be a causative factor in multifactorial syndrome implicated in the Gulf War and Tokyo terrorist attacks. While high dose results in seizure and death, low dose exposure may lead to autonomic imbalance and chronic cardiac pathologies. In the present study, echocardiography and electrocardiography were used to examine the late onset effects of a low dose sarin on cardiac structure and function in mice. Stress responsiveness of the hypothalamic pituitary adrenal (HPA) axis was also tested. Findings demonstrate changes consistent with a dilated cardiomyopathy, including left ventricular dilatation, reduced contractility, and altered electrophysiological and …

2,3,7,8-Tetrachlordibenzo-P-Dioxin Mediated Immune Suppression Through Interactions At The 3'Immunoglobulin Heavy Chain Regulatory Region Enhancers, David Harold Ellis

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2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD) is a potent toxin which inhibits the antibody response of B cells. The 3'IgHRR which is involved in transcriptional regulation of the heavy-chain polypeptide of antibodies is inhibited by TCDD. The hs1,2 enhancer region, isolated from the 3'IgHRR, is also inhibited while the isolated hs4 is activated by TCDD. This project sought to determine if that dichotomy in effects results from interactions at enhancer-specific binding sites for AhR, thought to mediate transcriptional effects of TCDD, and NFκB, a transcription factor involved in B cell activation. Here, I report a difference in the effect of TCDD on transcriptional activity …

Quantitative Structure-Activity Relationships For Organophosphates Binding To Trypsin And Chymotrypsin, Christopher Daniel Ruark

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Organophosphate (OP) nerve agents such as sarin, soman, tabun, and O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate (VX) do not react solely with acetylcholinesterase (AChE). Evidence suggests that a wide range of cholinergic-independent pathways are also targeted, including serine proteases. These proteases comprise nearly one-third of all known proteases and play major roles in synaptic plasticity, learning, memory, neuroprotection, wound healing, cell signaling, inflammation, blood coagulation and protein processing. Inhibition of these proteases by OPs was found to exert a wide range of noncholinergic effects depending on the type of OP, the dose, and the duration of exposure. Consequently, in order to understand …

Hyperbaric Oxygen In The Prevention Of Carbon Monoxide Induced Delayed Neurological Sequelae In Male Sprague Dawley Rats (Rattus Norvegicus), Chester P. Gut Jr.

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In Carbon Monoxide (CO) induced Delayed Neurological Sequelae (DNS) clinical signs develop 1 to 6 weeks after CO has cleared the body. The aim of this experiment was to develop a model of CO induced DNS which closely mimics "real world" conditions both in exposure and treatment. The model was challenged with hyperbaric or normobaric oxygen, or room air. Basic behaviors were measured by Open Field test on days 1, 7, 14 post exposure and treatment. No significant difference in behavior was observed between exposed and control animals or between treatment groups. Histological analyses showed no DNA or necrotic damage …

Tissue Specific Effects Of Adipose Stem Cells (Asc) In A Melanoma Tumor Environment, Drew Michael Nedderman

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This study determined the tissue-specific effect of adipose stem cells (ASC) within a melanoma environment for development of a cell-based melanoma therapy. Analysis included a subcutaneous B16-F1 melanoma model using thirty-one C57BL/6 wild-type mice. Melanoma xenografts were treated with cell-based therapies of CFDA-SE-labeled human fibroblasts HF20x (control), non-differentiated ndASC or hematopoietic-differentiated HdASC. No tumor regression was observed in presence of cell-based therapies, thus, the HdASC group demonstrated an increase in tumor growth accompanied with an up-regulated macrophage response, and increased angiogenesis. In addition, this group demonstrated a decrease in Melan-A tumor marker and interferon-γ expression suggesting that ASC-supported tumor angiogenesis …

Effects Of Direct Mechanical Ventricular Actuation On The Apoptotic Signaling Of A Failing Heart, Scott Kerns

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Cardiovascular disease accounts for more than 40% of all deaths in the United States (AHA-2004 report). A non blood contacting ventricular assist device (VAD) can be used to treat heart failure without the complications that arise from blood contacting VADs. This study used cellular markers of heart failure as indicators of heart function in an attempt to assess if direct mechanical ventricular actuation (DMVA) support lessened the impact of heart failure in rabbits. Cell signaling proteins were monitored using enzyme activity measurements and quantitative immunoblotting with antibodies against intrinsic and extrinsic apoptotic pathways during heart failure with and without DMVA …

In-Vitro Biocompatibility Of Silver Nanoparticles Anchored On Multi-Walled Carbon Nanotubes, Alicia Brooks Castle

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Silver (Ag) nanoparticles were anchored on the surface of three different multi-walled carbon nanotubes (MWNTs): Ag-MWNTs (pure carbon), Ag-MWNT-COx (carbonyl and carboxyl doped), and Ag-MWNT-CNx (Nitrogen-doped). The chemical reactivity of the nanotubes was MWNTs< COx-MWNTs< CNx-MWNT. Ag-NP anchored on the MWNTs had an average size corresponding to MWNTs 6.64nm +/- 2.25nm; CNx-MWNTs, 13.24nm +/- 3.94nm, and COx-MWNT/Ag 11.75nm +/- 4.65nm. Cellular function and immune response were evaluated to determine biocompatibility of the synthesized nanomaterials (NM) on the human keratinocyte cell line (HaCaT). Cellular assays revealed toxicity after 24h however, full cellular recovery was observed at 48h. These results suggest Ag nanoparticles anchored on MWNTs have the ability to interact with cells without disrupting cellular function.

Effects Of Chlorpyrifos-Oxon On Prohormone Convertase Enzyme Activity, Sean William Harshman

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Organophosphate (OP) compounds have been synthesized for various applications including medicine, pest control and weapons of terror. OPs irreversibly phosphorylate the active site of acetylcholinesterase, the primary target of toxicity, rendering it inactive causing a hypercholinergic state in the post synaptic cleft. However, very little is known about the effects of OPs on secondary targets of toxicity i.e. other serine hydrolases. The prohormone convertase (PC) enzymes, specifically PC1/3 and PC2, are serine hydrolases which are responsible for the processing of pro-opiomelanocortin in the anterior and intermediate lobes of the pituitary. To test the direct interaction between OPs and the PC …

Ischemic Stroke In Type Ii Diabetic Mice: Deregulation Of Sdf-1a/Cxcr4 Axis, Avik Das

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Type 2 diabetes mellitus is a major risk factor for ischemic stroke. Also diabetes is associated with poor outcome after stroke. Underlying mechanisms are however not fully understood. Alteration in the expression of the SDF-1a/CXCR4 axis, which is important for ischemic tissue repair, can be a probable cause. In this study, we have determined the expression of SDF-1a/CXCR4 in the brains of type II diabetic mice at basal and in response to ischemic stroke and have investigated a method for overexpression of SDF-1a in the brains of the diabetic mice. Adult male C57BLKS/J mice (db/db) of age 8 weeks were …

Effect Of Low-Dose Sarin Exposure On The Neurochemistry Of Different Brain Structures In Mice, Dhawal Pravin Oswal

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Sarin (GB) is a toxic organophosphate (OP) nerve agent that was released in the Gulf War and was used in terrorist attacks in Japan. People who survive such attacks exhibit various long-term effects including alterations in neuropsychological performances. It has also been hypothesized that the Gulf War Illness could be a result of low level exposure to OP's. In order to understand the effect of low dose exposure to GB on physiological and behavioral functions, we analyzed the levels of monoamines and their metabolites in different brain areas after exposure of mice to a sublethal dose of GB. Mice (male …

Impact Of Diabetes On Ace/Ace2 Balance And Angiotensin Ii Type 1 Receptor Expression In Db/Db Diabetic Mice, Malav Navinchandra Madhu

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Alterations in the renin-angiotensin system (RAS) are considered to be crucial for the development of diabetic complications like hypertension and nephropathy. Our previous work demonstrated role of AT1 receptors (AT1R) in the development of hypertension in db/db diabetic mice. The aim of this study was to test the hypothesis that there is upregulation of renal AT1R and imbalance in renal ACE/ACE2 homeostasis in db/db mice. In addition, we hypothesize that treatment with an anti-hyperglycemic or an AT1R blocker will correct this imbalance. Five week old control and db/db mice were housed in metabolic cages for 24 hour collection of urine. …

Characterization Of Infectivity And Pathogenesis Of Partially Reconstructed 1918 And Highly Pathogenic Avian Influenza Viruses In The Balb/C Mouse Model, John Allen Pyles

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Influenza viruses are consistently responsible for an average of 20,000 deaths and 114,000 hospitalizations per year. To a great extent, these viruses always stay one step ahead of the available vaccines and people's immunity year after year because they have the ability to either mutate part of their genetic material, or to be transmitted from one species to another. That same genetic variability explains why highly pathogenic influenza viruses emerge that cause great mortality over several countries resulting in pandemics. Highly pathogenic strains of influenza A virus have emerged occasionally in recent history, producing pandemics such as the one in …

Tcdd-Induced Modulation Of The Hs1,2 Enhancer Within The 3'Immunoglobulin Heavy Chain Regulatory Region, Tharu M. Fernando

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2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxin that inhibits immunoglobulin (Ig) gene expression in mice. Transcriptional regulation of the Ig heavy chain (IgH) involves the 3'IgH regulatory region (3'IgH RR). The murine 3'IgH RR consists of four enhancers (hs3A, hs1,2, hs3B, and hs4), which are homologous to the human 3'IgH RR enhancers (hs3, hs1,2, and hs4). In humans, a polymorphism of the hs1,2 enhancer, resulting in a varying number of tandem repeats of a 53 bp sequence, has been correlated with autoimmune diseases like IgA nephropathy and Celiac disease. The repeated sequence contains a kB and DRE binding site. Previous …

Identification Of Potential Protein Biomarkers Of Low Level Kidney Degradation, Christopher Lee Woolard

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The purpose of this research was the identification of potential dose-dependent and time-dependent serum protein biomarkers of low level kidney degradation in a rat model. Potential biomarkers were evaluated based on differential protein expression between control and dosed samples in rat serum. Proteins of interest demonstrated upregulation at a minimum 1.5 fold increase in protein concentration control versus dosed sample. In order to identify common biomarkers of kidney decrement, three nephrotoxins were chosen to target specific locations of the kidney: 1) D-Serine, which causes necrosis of the proximal straight tubules, 2) Puromycin, an antibiotic that degrades the Glomerular Basement Membrane …

Novel Neuroprotectants For Sarin Plus Cbdp Induced Convulsions, Kaushal V. Joshi

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Sarin, also known as Sarin (German agent B) is classified as a weapon of mass destruction. Sarin (O-isopropyl methyl phosphonofluoridate) is a highly toxic nerve agent originally produced for chemical warfare and has been used in terrorist activities. Sarin is an extremely potent acetylcholinesterase inhibitor with high specificity and affinity for the enzyme. High sarin doses causes death due to anoxia resulting from airway obstruction, weakness of the muscles of respiration, respiratory failure and convulsions. Current treatments are still not effective at protecting against long term effects following exposure. A current approach aims to counteract the increased glutamatergic and cholinergic …

Angiotensin At1 Receptor Blockade Protects The Brain From Ischemic Damage, Madhuri Penchikala

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Angiotensin (Ang) AT1 receptors are considered to play an important role in ischemic stroke via degenerative processes leading to cell death. Recent clinical and basic studies show that systemic blockade of Ang AT1 receptors reduces brain lesion in ischemic stroke. In this study we evaluated whether blockade of central Ang AT1 receptors protects the brain from ischemia and inflammation during ischemic stroke. Adult male C57BL/6 mice were divided into two groups for chronic intracerebroventricular (ICV) infusion of a selective Ang AT1 receptor antagonist, losartan (Los, n=18, 2 ug/hr) or isotonic saline (Con, n=20) using osmotic minipump. Twelve days post infusion, …

Neurodegeneration And Neuroinflammation In A Mouse Model Of Sarin Exposure, Molly Elizabeth Davidson

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Sarin is an organophosphorus (OP) ester chemical warfare agent (CWA) that has been used in past terrorist attacks. It remains a threat today because of its ease of manufacture and dispersal. Sarin acts by irreversibly inactivating acetylcholinesterase and interfering with neurotransmission by allowing acetylcholine to build up at neuro-effector junctions, where it continuously elicits a response. Symptoms of sarin toxicity include seizures, hypersecretions, respiratory distress and death in extreme cases. Previous studies on OP poisoning indicate that sarin exposure causes neurodegeneration and neuroinflammation in conjunction with seizure activity. In order to determine the mode of neuronal death and the extent …