VTechWorks Repository :: Browsing by Author "Ehrich, Marion F."

Browsing by Author "Ehrich, Marion F."

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Cefazolin Concentration in Surgically Created Wounds Treated with Negative Pressure Wound Therapy Compared to Surgically Created Wounds Treated with Nonadherent Wound Dressings
Coutin, Julia Viviana (Virginia Tech, 2014-06-25)
Our objective was to compare cefazolin concentrations in biopsied tissue samples collected from surgically created wounds treated with negative pressure wound therapy to those collected from surgically created wounds treated with nonadherent dressings. The study design was a prospective, controlled, experimental study. The animal population included 12 female spayed beagles. We hypothesized there would be a difference between the cefazolin concentrations of wounds treated with negative pressure wound therapy when compared to the cefazolin concentrations of wounds treated with nonadherent dressings. Surgical methods were as follows: Full thickness cutaneous wounds were created on each antebrachium (n=24). Following surgery, cefazolin (22 mg/kg) was administered intravenously to each of the dogs and continued every 8 hours during the study. The right wound was randomly assigned to group I or group II while the wound on the contralateral antebrachium was assigned to the other group. Group I wounds were treated with negative pressure wound therapy (NPWT) and group II wounds were treated with nonadherent dressings for 3 days. Dressings were changed and tissue biopsies obtained from wound beds at 24-hour intervals for both groups. Cefazolin wound tissue and plasma concentrations were measured by liquid chromatography mass spectrometry (LC-MS/MS). Blood samples for measuring plasma cefazolin concentrations were collected prior to biopsy sampling. At the time of surgery and at each bandage change, wound beds were swabbed and submitted for aerobic and anaerobic culture. Our results revealed that after initiating cefazolin treatment, wound tissue antibiotic concentrations between treatment groups were not significantly different at any sampling time. Similarly, after initiating cefazolin treatment, plasma cefazolin concentrations were not significantly different at any sampling time for individual dogs. We concluded that using a canine experimental model, NPWT treatment of surgically created wounds does not statistically impact cefazolin tissue concentrations when compared to conventional nonadherent bandage therapy
Characterization and Pharmacokinetics of Rifampicin Laden Carboxymethylcellulose Acetate Butyrate Particles
Casterlow, Samantha Alexandra (Virginia Tech, 2012-04-24)
Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is a common and potentially lethal infectious human disease. Rifampicin is a front line anti-tuberculosis drug usually prescribed in combination with isoniazid, pyrazinamide and streptomycin for a period of six to seven months. When given orally for the treatment of MTB, rifampicin exhibits low bioavailability. Recent attempts to increase bioavailability and decrease dosage of anti-tuberculosis drugs have focused on creating polymer coated rifampicin nanoparticles. The research effort presented in this thesis evaluates the formation, characterization and relative bioavailability of rifampicin loaded carboxymethylcellulose acetate butyrate (CMCAB) particles using two different formulation techniques. Multi inlet vortex mixer (MIVM) and manual spray drying techniques were used to form the rifampicin containing CMCAB particles. Characterization studies and analyses of particles revealed differences in particle sizes, shapes and drug loading between the different particle formulation techniques. In vivo pharmacokinetic studies in BALB/c mice indicate that a single dose of rifampicin laden CMCAB spray dried particle formulations are able to improve pharmacokinetic parameters including relative bioavailability of rifampicin compared to that of the free drug form at the same concentration.
Characterization of the Beta-2 Adrenergic Receptor Mechanism in Bovine Neutrophils, and Some Effects of Inflammatory Stimuli on its Function
LaBranche, Timothy Paul (Virginia Tech, 2005-04-12)
The bovine polymorphonuclear leukocyte (neutrophil) is a central component of the acute inflammatory response, and is capable of reacting to a myriad of pro-inflammatory chemical signals that have been characterized in the context of bovine respiratory disease (BRD). Human neutrophils and bovine macrophages are known to react to pro-inflammatory signals as well; however, they are also capable of responding to anti-inflammatory signals from the autonomic nervous system. In particular, activation of the beta2-adrenergic receptor on these cells decreases several aspects of inflammatory activity, including reactive oxygen species production, chemotaxis, degranulation, and inflammatory mediator production. Dysfunction of beta-adrenergic receptors is known to contribute to the pathophysiology of numerous diseases in both people and animals. For example, congestive heart failure, asthma, cystic fibrosis, atopic dermatitis, pheochromocytoma, myasthenia gravis, hypertension, and sepsis have all been linked to decreased beta1- / beta2-adrenergic receptor density (depending on the cell type) and / or uncoupling of the respective receptor from its effector enzyme, adenylyl cyclase. Dysfunction of the beta2-adrenergic receptor mechanism has also been described in pulmonary airway and vascular smooth muscle tissue from cattle, sheep, and rats exposed to Manheimia haemolytica, which provides insight into the pathophysiology of BRD. Despite the prominent role of the bovine neutrophil in the acute inflammatory stage of BRD, and despite the potential for dysfunction following excessive exposure to inflammatory stimuli, there are no reports that describe the presence of the beta2-adrenergic receptor on bovine neutrophils, nor function of the components responsible for its signal transduction cascade. Without complimentary work with bovine neutrophils, using data from human neutrophils to examine treatment options for the acute inflammatory stage of BRD is unrealistic. For this reason, the present dissertation proposed that 1) bovine neutrophils possess the beta2-adrenergic receptor mechanism, 2) components of the beta2-adrenergic receptor mechanism work in concert to increase bovine neutrophil adenosine 3,5-cyclic monophosphate (cAMP) levels and suppress superoxide anion production, and 3) the beta2-adrenergic receptor mechanism is dysfunctional following exposure to inflammatory stimuli. Using the nonselective beta1- / beta2-adrenergic receptor antagonist [3H]CGP-12177 we observed a maximum specific binding density (Bmax) value of 0.19 fmol per 100,000 bovine neutrophils. Although this value is approximately equal to what we observed with dairy cow neutrophils, human neutrophil Bmax values with this radioligand are anywhere from five to ten-fold greater, which suggests a significant species difference. We further defined the adrenergic receptor population on bovine neutrophils to be dominated by the beta2-subtype. Next, we characterized the function of beta2-adrenergic receptors by stimulating cAMP production with the beta2-adrenergic receptor agonist, terbutaline. The role of the beta2-subtype was confirmed when the terbutaline-mediated effect was negated by ICI-118,551, a beta2-adrenergic receptor antagonist. Also, the role of the phosphodiesterase enzyme in cAMP recycling in bovine neutrophils was illustrated, as the terbutaline-mediated rise in cAMP concentration was dependent upon phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine (IBMX). This study confirms the anti-inflammatory nature of the beta2-adrenergic receptor on bovine neutrophils by demonstrating the ability of terbutaline and IBMX to decrease superoxide anion production in a dose-dependent manner. The synthetic cAMP analog, 8-bromo-cAMP also decreased superoxide anion production, but the effect was time-dependent because of its need to diffuse across the cell membrane. Moreover, IBMX exaggerated the terbutaline-mediated effect on superoxide anion production, while cAMP exaggerated the IBMX-mediated effect on superoxide anion, demonstrating that the beta2-adrenergic receptor acts in concert with adenylyl cyclase, while the phosphodiesterase enzyme functions to decrease their signal. By increasing the dose of the inflammatory stimulant opsonized zymosan eight-fold, we were able to eliminate the ability of various concentrations of terbutaline and IBMX to reduce superoxide anion production. We sought to provide a more specific demonstration of this phenomenon by activating protein kinase C (PKC) via phorbol 12-myristate 13-acetate (PMA) administration. However, preincubation with PMA actually increased terbutaline-mediated cAMP production, in a dose and time-dependent manner. At this time, we cannot explain why increasing the dose of opsonized zymosan and PMA had opposite effects on beta2-adrenergic receptor mechanism function. The answer may reside in the many reported functions of PKC isoforms. Additional studies that identify the PKC isoform repertoire in bovine neutrophils may illustrate the potential for selective inhibition, and may lead to more specific identification and treatment of beta2-adrenergic receptor mechanism dysfunction. Also, it remains to be seen how the various components of the bovine neutrophil beta2-adrenergic receptor mechanism function in-vivo during the acute inflammatory stage of BRD.
Characterization, toxicity, and biological activities of organometallic compounds and peptide nucleic acids for potential use as antimicrobials
Ernst, Marigold Ellen Bethany (Virginia Tech, 2019-04-29)
Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised.
Characterizing Compensatory Effects of Silymarin on Gossypol Toxicosis in Lines of Chickens Divergently Selected for Humoral Immune Response
Blevins, Sarah (Virginia Tech, 2009-08-04)
Feed costs are approximately 70% of total production cost for poultry producers. Poultry diets in the United States generally consist of 2 grains: corn and soybean meal. In recent years, the cost of these grains has dramatically increased. Due to these price increases, producers seek alternative feeds that provide adequate nutrition, and are also more affordable than "traditional" grains. Cottonseed meal is one alternative that is both affordable and an excellent source of crude protein. However, cottonseed meal contains gossypol, a pigment toxic to chickens. This study had two main objectives. The first objective was to determine if silymarin, an extract from milk thistle, could offset or prevent gossypol toxicosis. The second objective was to determine if divergent selection for humoral immune response would have an impact on the ability of the chicken to cope with gossypol toxicosis. Two preliminary studies were conducted. One determined basal activities of liver detoxification enzymes at various ages. The other determined concentrations of gossypol and silymarin that should be added to the diet to elicit a response. The information gathered from the second preliminary study was used to conduct the final experiment. In the final experiment, chickens from each of 2 lines selected for humoral immunity were exposed to diets containing gossypol, silymarin, gossypol and silymarin, and a control. Humoral immunity had no impact on the ability of the chicken to cope with gossypol toxicosis. Silymarin did not alleviate gossypol toxicosis. Future studies will focus on using a lower gossypol concentration in the diet.
Circulatory, hormonal, and metabolic effects of arbutamine compared to exercise in persons with known or suspected coronary artery disease
Dorn, Karen Toft (Virginia Tech, 1994-10-05)
The purpose of this study was to test the hypothesis that arbutamine, a specific B₁-adrenergic agonist, will not cause different circulatory and physiologic effects than the less specific endogenous catecholamines released in response to an exercise stress test in persons with known or suspected coronary artery disease. Nine male subjects, mean age 66 years, completed symptom-limited arbutamine (ESA) and exercise (ETT) stress tests in a randomized cross-over study. The ESA delivery device controlled infusion rate to induce a graded heart rate increase of 8 bt min⁻¹. Heart rate, systolic blood pressure, diastolic blood pressure, rate pressure product, ST segment shift, and specimens for epinephrine, norepinephrine, dopamine, cortisol, insulin, glucagon, glucose, free fatty acids, glycerol, and lactate were collected at baseline, immediate post-stress, and 10, 30, and 60 minutes post-stress. The research hypothesis was rejected. Repeated measures analysis of variance for each measure demonstrated a significant (p ≤ .05) time treatment interaction in heart rate, systolic blood pressure, rate pressure product, insulin, glucagon, glycerol, free fatty acid, and lactate responses and a significant time effect for cortisol response. Circulatory differences included higher systolic blood pressure and rate pressure product responses for ETT than ESA and a more rapid recovery of circulatory variables following ETT. Metabolic differences were due to higher free fatty acid and glycerol responses for ESA than ETT and a slower recovery of these metabolites and lactate following ESA. Hormonal differences included an earlier and greater magnitude rise in insulin response for ESA than ETT. There were no differences (p ≤ .05) by treatment, time, or time treatment interaction for diastolic blood pressure, ST segment shift, catecholamines, or glucose. In conclusion, arbutamine caused different circulatory and physiologic effects consistent with differences in adrenergic receptor activity. Arbutamine caused substantial B₁₊₂ agonist effects on hormonal and metabolic responses in cardiotonic doses employed clinically for diagnostic stress testing and may impact clinical interpretation of stress test results.
Comparative evolution of mipafox-induced delayed neuropathy in the rat and hen
Carboni, Deborah Ann (Virginia Tech, 1993-08-09)
The group of chemicals designated organophosphorus compounds have had a significant impact on modern life, including use as pesticides, industrial plasticizers and chemical warfare agents. Exposure to certain organophosphates produces a delayed degeneration of the longest and largest nerve fibers, including those of the ascending and descending tracts of the spinal cord, a condition termed organophosphorus ester-induced delayed neuropathy (OPIDN). Recorded incidents of such an effect in humans have led to research regarding this neurological disease. Among the OPIDN-inducing agents is mipafox, an organophosphate insecticide, the compound we chose to employ in our studies. Although the hen is the primary experimental model in the safety assessment of organophosphates, current research has suggested that the rat may have some validity as an experimental model. We examined the sequential neuropathic effects of a single dose of mipafox (30mg/kg) in rats and hens on a comparative basis to determine the better experimental model.
Comparison of the Effects of Deracoxib, Buffered Aspirin, and Placebo on the Gastric Mucosa of Healthy Dogs
Sennello, Kathleen Ann (Virginia Tech, 2005-04-04)
This study tested the hypothesis that administration of deracoxib, a cyclooxygenase-2 specific (COX-2) inhibitor, would result in lower gastric lesion scores than administration of buffered aspirin and gastric lesion scores similar to placebo when administered to healthy dogs for 28 days. Twenty-four, healthy, random source dogs were divided into three groups. Group I received buffered aspirin, 23.6 mg/kg PO q 8h, group II received deracoxib, 1.6 mg/kg PO q 24h and placebo twice daily PO q 8h after deracoxib administration, and group III received placebo PO q 8h. Gastroscopy was performed on days -7, 6, 14, and 28 of treatment. Four regions of the stomach (pylorus, incisura, cardia, and body) were evaluated separately and lesions scored on a scale of 1 (mucosal hemorrhage) to 12 (perforating ulcer) by an observer unaware of which treatments the dogs received. Dogs were observed every 8 hours for vomiting, diarrhea and anorexia. Feces were scored from 1-5 (scores <4 were considered diarrhea). Lesion scores for each group, at each location, and total scores, at each time period, were evaluated for the effects of time and treatment using a Kruskal-Wallis test. Total dog days of vomiting and dog days of diarrhea in each group were compared using a Wilcoxon rank sums test. Significance was determined at p<0.05. Significantly higher median total gastric lesion scores were found in the aspirin group compared to the deracoxib or placebo groups on days 6, 14, and 28. There were no significant differences in median total gastric lesion scores between the deracoxib or placebo groups at any time during the study. There was no location effect on gastric lesion scores and there was no significant change in gastric lesion scores over time in any of the groups during treatment. Significantly more dog-days of vomiting occurred in the aspirin group as compared to the deracoxib group. No significant differences were found between groups for dog-days of diarrhea. In this study, the administration of deracoxib to healthy dogs resulted in significantly lower gastric lesion scores compared to dogs receiving aspirin and lesion scores similar to those receiving placebo.
Development of a model cell culture system in which to study early effects of neuropathy-inducting organophosphates
Nostrandt, Amy Carol (Virginia Tech, 1991)
Certain organophosphorus (OP) compounds produce a delayed neuropathy in man and susceptible animal species after early inhibition and aging of the enzyme, neurotoxic esterase (neuropathy target esterase, or NTE). In this study, the human neuroblastoma cell line, SY-5Y, was examined for its potential to serve as a nonanimal model for the study of the early effects of neuropathy-inducing OPs. For these investigations, the time course of inhibition and aging of NTE after toxicant treatment in neuroblastoma cells was compared to that in brain tissue from the adult chicken, which is the recognized animal model for organophosphorus ester-induced delayed neuropathy (OPIDN). Concentrations of toxicants to be used for treating neuroblastoma cells were determined after observing viability of treated differentiated SY-SY cells incubated for 24 hours with a range of concentrations of an OP (mipafox) that induces neuropathy, an OP (paraoxon) that does not induce neuropathy, a carbamate (aldicarb), a neurotoxicant (β, β’-iminodipropionitrile, or IDPN) that acts by a different mechanism than the OPs, and a cholinergic agonist (carbachol). Treatment concentrations were chosen that caused less than 30% loss of viability over the 24 hour period. The time course and extent of detrimental effects of mipafox, which induces OPIDN in hens, and the carbamate, aldicarb, on NTE were similar in the SY-5Y cells to those observed in homogenized chicken brain tissue after the same treatments. Mipafox produced rapid inhibition and aging of NTE, with maximal effects occurring within 10 minutes of exposure. Aldicarb inhibited NTE but did not age the enzyme. Instead, spontaneous reactivation was observed both in SY-5Y cells and in brain tissue. None of the other negative control compounds (paraoxon, IDPN, carbachol) affected NTE activity in either SY-5Y cells or chicken brain tissue. To determine if the neuroblastoma cells could be used to study early events that could lead to modification of OPIDN, NTE inhibition and aging were determined in the differentiated SY-5Y cell line after mipafox was removed. Removal of mipafox from the cell culture medium at 5 minutes after exposure, or earlier, resulted in essentially no NTE inhibition or aging. NTE inhibition and aging were also determined after treatment of the SY-5Y cells with the neuropathy-inducing OP, mipafox, and representatives of 2 classes of compounds (carbamates and calcium channel blockers) previously demonstrated to modify OPIDN in hens. The modifiers (aldicarb and verapamil) were used as a 5 minute pre-treatment, simultaneous treatment, and a 2 minute post-treatment. Significant prevention of most of mipafox-induced NTE inhibition and aging was observed. Effects on NTE inhibition and aging in differentiated SY-5Y cells after each mipafox-aldicarb combination and mipafox-verapamil combination of treatments were similar to those in chicken brain homogenate. These results indicated that both aldicarb and verapamil protected NTE against the early biochemical effects of mipafox that are thought to initiate OPIDN in vivo. The temporal relationship of NTE inhibition and aging to other detrimental effects on neuroblastoma cells was assessed by the capability of mipafox to cause changes in free intracellular calcium ion concentration, measured using fluorescent calcium probes, and by its capability to alter cell morphology as assessed by phase contrast microscopy. NTE inhibition and aging preceded these changes. Capability to inhibit activity of the neural enzyme, acetylcholinesterase, was also determined. The results of these studies indicate that the SY-5Y model system shows promise for use in the determination of initial mechanisms contributing to the development of organophosphorus-induced delayed neuropathy.
Development of nanoparticle based nicotine vaccines for smoking cessation
Hu, Yun (Virginia Tech, 2015-06-15)
Cigarette smoking is prevalent worldwide and has consistently been the top preventable cause of many serious diseases., which result in huge mortality, morbidity, and economic loss, in recent decades. In recent years, nicotine vaccines that can induce production of nicotine specific antibodies in human have emerged as a promising medicine to treat tobacco addiction. In the past decade, there have been numerous nicotine vaccine candidates evaluated in human clinical trials, including NicVaxNicVAX®, TA-NICTA-NIC®, Nic002NIC002®, NiccineNiccine®, and SEL-068SEL-068®. . However, traditional nicotine vaccine designs haves many disadvantages, including low immunogenicity, low specificity, difficulty in integration of molecular adjuvants, and short immune response persistence. To overcome the above limitations, in this study, various nanoparticle-based vaccine delivery systemsvaccine componentss have been developed and evaluated as potential delivery vehicles for vaccines against nicotine addiction. Firstly, a nicotine vaccine was synthesized by conjugating bovine serum albumin (BSA)-nicotine complex to the surface of nano-sized cationic liposome. Significantly higher anti-nicotine antibody titer was achieved in mice by liposome delivered nicotine vaccine compared with nicotine-BSA vaccine. Secondly, a novel nanoparticle (NP)-based delivery platform was constructed by incorporating a negatively charged nanohorn into cationic liposome to improve the stability of liposome and reduce nanoparticle flocculation. Subsequently, nicotine vaccine was constructed by conjugating nicotine-BSA complex to the surface of the nanohorn supported liposome (NsL). Marked improvement in stability in vitro and significant increase in titer of anti-nicotine antibodies were detected in nanohorn supported liposome ( NsL) delivered vaccine than liposome delivered vaccine. In addition, NsL nicotine vaccine exhibited good safety in mice after multiple injections. Thirdly, lipid- poly(lactic-co-glycolic acid) (PLGA) hybrid NPs were constructed as vaccine delivery system. due to the fact that nanohorn is not currently approved for clinical use, we substituted the nanohorn with poly(lactic-co-glycolic acid) (PLGA) nanoparticles and constructed PLGA-lipid hybrid nanoparticles. Preliminary results showed that PLGA-lipid hybrid NPs nanoparticles exhibited improved stability, better controlled release of antigens, as well as enhanced uptake by dendritic cell (DC). A lipid-PLGA hybrid NPnanoparticle was also developed that was structurally responsive to low pH challenge. The lipid shell of the hybrid nanoparticle was rapidly disintegrated under a low pH challenge, which resembles the acidic environment of endosomes in DCsdendritic cells. The hybrid NPs exhibited minimal antigen release in human serum at physiological pH, but a faster release of antigen from this NP compared to non-pH sensitive NPs was observed in DC. In the final study, hybrid NPnanoparticles with various cholesterol concentrations were constructed. Slower and more controlled release of antigens in both human serum and phosphate buffered saline were detected in nanoparticles with higher cholesterol content. However, nanoparticles containing higher cholesterol showed poorer stability due to increase fusion among NPnanoparticles. It was later found that PEGylation of NPs can effectively minimize fusion caused size increase after long term storage, leading to improved cellular uptake. The findings from this study on the nanohorn-lipids based nicotine vaccine as well as lipid-PLGA hybrid NPs may provide solid basis for future development of lipid-PLGA based nicotine vaccine.
Early Effects of Organophosphate Compounds on In Vitro Intracellular Signaling and Levels of Active Neurotrophin Receptors, and on In Vivo Neurotrophin Concentrations
Pomeroy-Black, Melinda J. (Virginia Tech, 2005-10-14)
Organophosphorus (OP) compounds are found in household pest control products, plastics, and petroleum. Due to the neurotoxic nature of OP compounds, exposure can cause both acute and delayed symptoms, including organophosphate-induced delayed neuropathy (OPIDN). This syndrome is characterized by Wallerian-like degeneration of nerves in the central and peripheral nervous system after exposure to neuropathic OP compounds. There are many questions surrounding the mechanisms of the onset of OPIDN, including possible alterations in proteins associated with neuronal maintenance and repair. This dissertation investigated the changes in levels of neurotrophins in vivo and how in vitro levels of neurotrophin receptors and their downstream signaling cascades are affected after exposure to OP compounds. We also characterized the molecular weight of a soluble factor responsible for inducing neurite outgrowth in vitro after in vivo exposure to a neuropathic OP compound. We evaluated in vivo endpoints using enzyme-linked immunosorbant assays. Results indicated that nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are found in chicken spinal cord but do not increase as a result of exposure to neuropathic OP compounds. This study also noted that NGF, BDNF, and NT-3 concentrations were not altered after exposure to a non-neuropathic OP compound. We evaluated in vitro endpoints using Western blots, ultrafiltration, and digital morphometry. These studies revealed that activated forms of high-affinity and low-affinity neurotrophin receptors are present after OP compound exposure, that the ratio of these two receptors to each other is stable after OP compound exposure, and that the activated form of the low-affinity receptor, which can lead to apoptosis, was present in greater levels than the activated form of the high-affinity receptor. Furthermore, OP compound exposure resulted in time-dependent changes of protein levels central to the mitogen-activated kinase and phosopholipase C-gamma intracellular pathways. Changes in a third pathway, the protein kinase C pathway, were dependent on the concentration and type of OP compound. Finally, in vitro neurite length was not affected by the type of OP compound administered in vivo or when a whole protein fraction was separated by molecular weight. This research has revealed in vivo consequences and early effects on intracellular protein and activated neurotrophin receptor levels after OP compound exposure. These early effects may contribute to the delayed development of neurotoxic effects associated with OP compound exposure.
The Effect of Organophosphate Exposure on Neocortical, Hippocampal and Striatal Monoamines: A Potential Substrate for Chronic Psychiatric, Cognitive and Motor Dysfunction
Lewis, Mary Catherine (Virginia Tech, 2003-08-20)
Depression and other mood disorders, as well as cognitive and motor dysfunction have been linked with changes in monoamine levels in the brain. Environmental acetylcholinesterase (AChE) inhibitors, such as organophosphate insecticides (OPs), have also been shown to induce these problems. This study investigated whether insecticide-induced AChE inhibition, induced by chlorpyrifos (CPS), may contribute to the types of forebrain monoaminergic alterations associated with psychiatric, cognitive and motor dysfunction. Increased synaptic ACh, resulting from CPS-induced AChE inhibition, may alter the synthesis or release of monoamines through prolonged action of ACh on monoaminergic neurons that contain ACh receptors. Adult, male Sprague-Dawley rats were subjected to a single subcutaneous dose of CPS or corn oil vehicle. Brains were rapidly removed and the frontal cortex, hippocampus and striatum were bilaterally dissected on ice. These three regions from one side were assayed for AChE activity, while those from the opposite side were processed for high performance liquid chromatography with electrochemical detection (HPLC-ED) analysis of monoamine neurotransmitters and their metabolites. In the initial, exploratory experiment, inhibition of AChE activity was 66.8% in the frontal cortex, 43.8% in the hippocampus and 46.9% in the striatum, 7 days after a 60mg/kg dose of CPS. No significant differences in concentration of monoamine neurochemicals were observed between vehicle control and CPS-treated groups in either the hippocampus or striatum. However, in the frontal cortex of the CPS-treated rats there was a significant increase in median dihydroxyphenylacetic acid (DOPAC) concentration (P=0.019) and a very strong statistical trend toward increased dopamine (DA) concentration (P=0.0506). The second experiment examined the time course of AChE inhibition produced by a higher dose (200mg/kg) of CPS and how monoamine levels changed in conjunction with this pattern of AChE inhibition. Percent inhibition of AChE activity in CPS-treated animals, at 4, 14 and 21 days post-exposure was 77.0%, 86.6% and 81.9% in the frontal cortex, 86.1%, 85.9% and 83.2% in the hippocampus and 90.1%, 89.8% and 85.5% in the striatum. No significant differences in monoamine neurochemicals were observed between vehicle control and CPS-treated groups in either the hippocampus or striatum. A statistical trend toward a decrease in serotonin (5-HT) was seen in the frontal cortex at 14 days (P=0.0753) following CPS exposure. A very consistent, yet non-significant pattern of an increase in monoamines at 4 days post-CPS was observed in all instances, except for 5-hydroxyindoleacetic acid (5-HIAA) in the striatum. Therefore, the final experiment employed a more powerful design to focus on monoamine levels during, or shortly after, the change in AChE activity that rapidly follows exposure to 200mg/kg CPS. This experiment also employed a behavioral analysis on the day of sacrifice to assess the presence or absence of clinical signs of toxicity associated with this dose. Of the 30 CPS-treated rats, only 1 animal displayed a single behavioral sign of cholinergic poisoning. Percent inhibition of AChE activity at 2 and 4 days after treatment was 81.4% and 79.4% in the frontal cortex, 53.4% and 83.5% in the hippocampus, and 80.5% and 87.8% in the striatum. No significant changes in monoamine neurochemicals were observed between vehicle control and CPS-treated groups in either the frontal cortex or hippocampus. However, a significant increase in DOPAC (P=0.0285) in the striatum, 2 days after CPS treatment, was observed. In addition, a strong statistical trend toward decreased striatal 5-HT (P=0.0645) was reported 4 days after CPS treatment. The only significant correlation between AChE activity and monoamine concentration was observed for 5-HIAA in the striatum of CPS-treated, 2 day survivors (P=0.0445). However, it was of low magnitude (r=0.525, r2=0.276). CPS has a limited capacity to produce changes in monoamine neurotransmitters and/or their metabolites in the frontal cortex and striatum of the mammalian brain. These changes are primarily seen in the dopaminergic system. Alterations of monoamines do not appear to be strongly associated with incident levels of AChE inhibition. The biological implication of the limited OP induced changes in central monoamines remains significant, as changes in monoamines in the CNS nervous system have been linked to psychiatric, cognitive and motor dysfunction.
Effects of Methylmercury Exposure on the Immune and Neurological Responses of Mice to Toxoplasma gondii Infection
King, Marquea D. (Virginia Tech, 2002-06-21)
Toxoplasma gondii is a protozoan parasite that causes life-threatening disease in congenitally infected infants and immunocompromised patients, such as those inflicted with AIDS. Toxoplasmic encephalitis (TE) is a common presenting condition in an AIDS infection. People become infected with T. gondii by ingesting tissue cysts in undercooked meats or by ingesting oocysts excreted by cats. Methylmercury (MeHg) is a well-documented neurotoxicant that accumulates in the brain and causes severe mental and visual dysfunction, including chronic encephalopathy. Consumption of contaminated fish, grains, and seeds are common sources of human exposure to methylmercury. Studies from our laboratory suggest that oral exposure to a single high dose of 20 mg/kg MeHg does not increase the susceptibility to acute toxoplasmosis in CBA/J mice. Therefore, we further investigated endpoints associated with immunotoxicity and neurotoxicity in 6-week old, female CBA/J mice exposed to both MeHg and T. gondii during a chronic T. gondii infection. We examined both single and multiple doses of MeHg exposure in a chronic parasitic infection model. In the single high dose study, four groups of six-week-old, female CBA/J mice were either fed 25 T. gondii tissue cysts of the ME-49 strain or given vehicle. Six weeks later, two out of the four groups (T. gondii and vehicle control) were orally gavaged with a single dose of 20 mg/kg body weight of MeHg and sacrificed seven days post exposure. Experiments from the multiple MeHg dose study were performed under similar conditions with the same number of groups and dosed by oral gavage with 8 mg/kg body weight of MeHg on days 0, 2,4,7,10,13. These mice were sacrificed on day 17 or 18 after initiating MeHg exposure. Flow cytometry following exposure to a single dose of MeHg in mice with a chronic T. gondii infection revealed significant changes (P < 0.05) within the T cell subpopulation percentages caused by exposure to MeHg. For example, the thymic CD4+CD8+ T cell subpopulations were increased (P <0.05). However, MeHg had no significant effect on the CD4+CD8-, CD4-CD8+, or non-T cell subpopulations in the spleen. Furthermore, MeHg increased splenic cellularity and spleen-to-body-weight ratios with or without a concurrent T. gondii infection. MeHg also caused a significant decrease in mouse body weight. There was a significant (P <0.05) increase in brain tissue cyst counts within the group exposed to both MeHg and T. gondii (16 ± 4, mean ± SE, n=7) versus T. gondii alone (4 ± 1, n=8). Histopathological examination demonstrated that the brain was affected, as lesions, gliosis, and meningitis were notable in mice given T. gondii. Exposure of mice to multiple doses of MeHg also resulted in effects on the immune system of CBA/J mice with and without chronic toxoplasmosis. Total cellularity and numbers of CD4+CD8+, CD4+CD8-, CD4-CD8+, and CD4-CD8- T-cell subpopulations show a marked decrease in number in the thymus, while total cellularity was also decreased in the spleen following concurrent exposure to T. gondii and MeHg. Flow cytometric examination of lymphocyte populations (CD4+ and CD8+ lymphocytes) in the spleen and thymus demonstrated differences from control in the groups exposed to T. gondii and MeHg. Histopathological examination did not reveal any significant lesions. The data from experiments in which single or multiple doses of MeHg were given to mice with a chronic T. gondii infection indicate that concurrent exposure, to both MeHg and T. gondii, dependent on dose and time of exposure had notable effects, especially on the immune system (Supported by NIH Grant F36GM20301).
The Effects of Neuropathy-Inducing Organophasphate Esters om Chick Dorsal Root Gangli Cell Cultures
Massicotte, Christiane (Virginia Tech, 2001-09-09)
Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN.
Effects of organophosphate esters on blood vessels: a physiological, pharmacological, and histological assessment of involvement in organophosphorus-induced delayed neuropathy (OPIDN)
McCain, Wilfred C. (Virginia Tech, 1994)
The contribution of the cardiovascular system. to organophosphate-induced delayed neuropathy (OPIDN) was examined using in situ and in vitro models for demonstration of response to vasoactive agents (e.g., the cholinergic agonist, acetylcholine; the α1 agonist, phenylephrine; and the β2 agonist, salbutamol). These responses were compared before and 1, 3, 7, and 21 days after hens were administered cyclic phenyl saligenin phosphate (PSP, 2.5 mg/kg i.m.), an OP that induces OPIDN but does not significantly inhibit acetylcholinesterase activity, and paraoxon (PXN, 0.1 mg/kg i.m.), an OP that inhibits acetylcholinesterase activity but does not induce OPIDN. The capability of verapamil, a calcium channel blocker, to attenuate these responses was examined, as this agent ameliorates OPIDN. For the in situ study, the ischiadic artery was cannulated and alterations in pressure measured at a constant flow used to indicate changes in vascular resistance. Changes in vascular resistance in response to acetylcholine, phenylephrine, and salbutamol that were different from those in control and PXN-treated hens were noted 1 and 3 days after administration of PSP. These changes were attenuated in hens given PSP and verapamil. Vascular segments from the ischiadic artery were used to provide an in vitro model to determine if OPs caused direct vascular damage that was responsible for effects seen in the in situ model. In the in vitro model, however, responses of PSP and PXN were similar and not modified in vascular segments from hens given verapamil as well as the OPs. This indicated that the contribution of the cardiovascular system to OPIDN was due to more than a direct effect on relatively large caliber vessels. The contribution of the cardiovascular system to OPIDN also did not appear to relate to morphological changes induced by administration of OPs, as no changes in vascular morphology were noted. An OP-induced effect that could contribute to vascular effects noted are levels of plasma catecholamines. These levels were altered in hens given PSP or PXN, with increases seen after administration of PSP and decreases seen after administration of PXN. These alterations in plasma catecholamine levels were attenuated in hens given both verapamil and OP.
Effects of Prednisone or Prednisone with Ultralow-Dose Aspirin on the Gastroduodenal Mucosa of Healthy Dogs
Graham, Allison Heather (Virginia Tech, 2009-03-20)
This study tested the hypothesis that administration of immunosuppressive doses of prednisone in conjunction with ultralow-dose aspirin (0.5 mg/kg/day) would result in gastroduodenal lesion scores similar to those found in dogs administered only immunosuppressive doses of prednisone, but that the gastroduodenal scores from both of these treatment groups would be significantly higher than placebo when administered to healthy dogs for 27 days. Eighteen healthy adult purpose-bred dogs were divided randomly into three groups. Group I received placebo capsules and placebo suspension, Group II received prednisone capsules (mean 2.3 mg/kg, range 2.0-2.4) and placebo suspension, and Group III received prednisone capsules (mean 2.3 mg/kg, range 2.3-2.5) and aspirin suspension (0.5 mg/kg) by mouth once daily for 27 days. Gastroduodenoscopy was performed on days -7 (baseline), 5, 14, and 27 of treatment. Four regions of the stomach (angularis incisura, body, pylorus, and cardia) and the proximal descending duodenum were systematically scored on a scale of 1 (normal) to 11 (perforating ulcer) by an experienced observer who was blinded to the treatment groups and clinical signs of each subject. Dogs were observed every 8 hours for vomiting, diarrhea, and inappetence. Feces were scored on a scale of 1-5 with diarrhea defined as a fecal score <4. Lesion scores for each group, at each location, and total scores, at each time period were evaluated for the effects of time and treatment using a Kruskal-Wallis test. Total dog days of vomiting and dog days of diarrhea in each group were compared using a Wilcoxon rank sums test. Significance was determined at p<0.05. There were no significant differences in median total gastric lesion scores between any of the groups at any time during the study. There was no location effect on regional gastroduodenal lesion scores and there was no significant change in gastroduodenal lesion scores over time in any of the groups during treatment. Significantly more dog-days of diarrhea occurred within the prednisone and aspirin group during the experimental period (Period 2) in comparison to Period 1. However, no significant differences were found between any of the groups for dog-days of vomiting, diarrhea or inappetence at any time in the study.
Effects of Quaternary Ammonium Disinfectants on Mouse Reproductive Function
Melin, Vanessa Estella (Virginia Tech, 2015-07-25)
Quaternary ammonium compounds (QACs) are antimicrobial disinfectants commonly used in commercial and household settings. While these compounds have been used for decades, reproductive toxicity has not been thoroughly evaluated. Extensive use of QACs results in ubiquitous human exposure to potentially toxic compounds. Reproductive toxicity of two common QACs, alkyl dimethyl benzyl ammonium chloride (ADBAC) and didecyl dimethyl ammonium chloride (DDAC), was investigated to determine gender-specific toxicity with an emphasis on male reproductive function. Breeding pairs of mice exposed for six months to ADBAC+DDAC exhibited decreases in fertility and fecundity, with fewer pregnancies and decreased numbers of pups over a six month period. Females proceeded through significantly fewer estrus cycles, and both ovulation and implantation rates were reduced. Males exhibited declines in both sperm concentration and motility. Male reproductive toxicity was further assessed in a series of in-vitro and in-vivo experiments. ADBAC+DDAC were cytotoxic to testicular Sertoli cells in culture at concentrations greater than or equal to 0.0005%. Changes in blood-testis-barrier integrity (BTB) were observed at 0.01% ADBAC+DDAC using a two-compartment culture system that measures transepithelial electrical resistance (TER). Sertoli cell cytotoxicity correlated with decreased TER at ADBAC+DDAC concentrations above 0.001%. In-vitro fertilization capacity of epididymal sperm was reduced in males given a 10-day rest period following ADBAC+DDAC exposure. Multigenerational changes in sperm parameters and in mRNA expression of enzymes involved with epigenetic modifications were evaluated across three generations. Sperm concentration and motility were reduced in F0 males exposed directly to ADBAC+DDAC. In F1 males, sperm concentration was increased and motility decreased, while there was no change in the F2 progeny. Genes involved in epigenetic modifications were altered in the exposed F0, with upregulation of two histone acetyltransferases (Hat1 and Kat2b) and downregulation of one lysine-specific demethylase (Kdm6b). F1 and F2 generations were not different from controls except for downregulation of the methyltransferase Dnmt1 in F1 progeny. The reproductive toxicity of ADBAC+DDAC identified in these studies, particularly to the male, compels further investigation into the potential effects that these compounds may have on human reproduction.
Evaluation of ceftiofur sodium as a chemotherapeutic agent in grass carp (Ctenopharyngodon idella)
Somjetlertcharoen, Amornchai (Virginia Tech, 2001-03-23)
Ceftiofur sodium, a third generation cephalosporin, was studied to determine the potential of this drug as an alternative bacterial therapeutic agent for the aquaculture and ornamental fish industry. Grass carp, Ctenopharyngodon idella have been selected as the fish model for this study since they are a good representative for both foodfish and ornamental fish and are one of the major species grown worldwide. Pharmacokinetics of ceftiofur sodium after various routes of administration, histopathologic observations to detect possible toxic effects on the tissues involved in its metabolism and excretion, and the effects on the non-specific immune response were investigated in grass carp. For the pharmacokinetic studies, ceftiofur sodium was administered a single time to grass carp by four different routes : intracardiac (IC), intraperitoneal (IP), intramuscular (IM) and oral (PO) at a dosage of 8 mg/kg body weight. Serial blood samples were obtained and plasma samples were analyzed by high performance liquid chromatography for ceftiofur (as measured its metabolite, desfuroylceftiofur (DFC) and DFC-related metabolite concentrations). Disposition pharmacokinetic data were best described by a two compartment open model for IC and by a non-compartment model with no lag time for IP and IM administrations. Oral absorption of ceftiofur was not observed in this species. Following IC, IP and IM ceftiofur sodium administration, the final elimination half-lives, maximum plasma concentration, time to reach maximum concentration, volume of distribution and plasma clearance were 0.38, 0.45 and 13.86 hours ; 157.09, 31.54 and 8.86 mg/ml ; 0, 0.25 and 0.5 hours ; 0.09, 0.17, 0.53 l/kg ; and 0.21, 0.26, 0.26 ml/min.kg, respectively. Desfuroylceftiofur metabolite was highly bound with plasma protein at pH 7.0 and 8.0. For the histopathological studies, a single intramuscular dose of ceftiofur sodium at three different concentrations, 8 (1X), 40 (5X) and 80 (10X) mg/kg was administered to separate groups of grass carp for evaluation of the potential toxicity to major tissues involved in metabolism and excretion of this drug. These included the anterior kidney, posterior kidney, liver, and spleen. After 48 hours, lesions were seen in the posterior kidney at the highest dose of ceftiofur (10X). Morphological alterations observed microscopically included increased number of renal tubules, tubular necrosis and infiltration of inflammatory cells. No adverse effects on the glomeruli were observed at any concentration of the drug. For the immunotoxicity studies on the non-specific immune response, dosages of either 8 or 40 mg/kg body weight were administered intramuscularly. After 24 and 48 h, leukocyte number, phagocytic ability and H2O2 production were examined in the cells of the pronephros. The results showed that neither dosage had an effect on the number of leukocytes in the pronephros. Phagocytosis was also not significantly altered at either dosage in macrophages from the pronephros. Hydrogen peroxide production was not altered in the pronephros of fish dosed at 8 mg/kg, while at a dosage of 40 mg/kg, H2O2 production was significantly increased. In summary, ceftiofur sodium has potential as an efficacious chemotherapeutic agent for controlling bacterial infection in brood stock and ornamental fish at the recommended dose of 8 mg/kg. A dose as high as 40 mg/kg can be use with careful consideration. This dosage may not directly injure the posterior kidney but it may affect the non-specific immune response of the fish.
Evaluation of the Role of Astrocyte Glutamate Transport and of Synaptic NMDA Receptor Subtype Representation in the Pathogenesis of PTSD
Cotrone, Thomas Steven (Virginia Tech, 2017-06-22)
Post-traumatic stress disorder (PTSD) is a psychological disorder that can cause great social/economic hardship. Progress towards treating PTSD has been slow due to a lack of understanding of its pathogenesis. This dissertation aimed to address this issue by investigating the involvement of the astrocytic glutamate reuptake transporter, GLT-1, and regional differences in expression of NMDA receptor subtypes in the development of a rat model of PTSD. We hypothesized that impaired astrocytic glutamate reuptake inhibits long-term memory processes, and that concurrent presence of glucocorticoids (GCs) during situational trauma selectively inhibits fear extinction memory processes in the prefrontal cortex, but not of conditioned fear memory processes in the amygdala, due to differences between these brain regions in expression of NMDA receptor subtypes. The effect of GLT-1 manipulation was studied in vivo. Utilizing the Single Prolonged Stress (SPS) model of PTSD, rats were either exposed to SPS or not. Within these groups, rats were administered a saline sham, a GLT-1 facilitator (ceftriaxone (CEF)), or a GLT-1 inhibitor (dihydrokainic acid (DHK)). Using Classical Fear Conditioning (CFC) and Fear Extinction (EXT) paradigms, retention of fear extinction memories was measured to determine the effect of GLT-1 manipulation on SPS-induced behavior (i.e., impaired fear extinction retention). From the brain of each rat, the amygdala, hippocampus, and prefrontal cortex (PFC) were collected and expression of GLT-1, p-CREB (a molecular indicator of long-term memory), and glucocorticoid receptor (GR, a molecular indicator of a PTSD-like state) were quantified. Analysis of the behavioral data showed that SPS exposure alone reduced the retention of extinction memories, but CEF and DHK both eliminated this effect. Analysis of the brain tissues revealed that SPS induced an increase in GR expression in the hippocampus. SPS also increased GLT-1 expression, but not p-CREB, in the PFC and amygdala. To evaluate the involvement of regional differences in NMDA receptor subtype expression ex vivo, tissue sections of amygdala, hippocampus, and PFC were taken from SPS and non-SPS exposed rats. Synaptic transmission was stimulated in these tissues using bicuculline in the presence of glucocorticoids, NVP-AA077 (a NR2A NMDA receptor subtype inhibitor), or Ro-25 (a NR2B NMDA receptor subtype inhibitor). P-CREB was measured in the tissues treated with GCs to determine if GCs exert greater inhibition of long-term memory in the PFC (a region reported to express high NR2A) than in the amygdala (a region reported to express high NR2B). P-CREB was also measured in the tissues treated with NVP or Ro-25 to determine if these reported receptor profile differences could be demonstrated, and if they changed following SPS exposure. Contrary to the stated hypothesis, analysis of non-SPS exposed rats revealed that GCs, NVP, and Ro-25 decreased p-CREB in all three regions with no differences between regions. However, in the SPS exposed group, p-CREB was not decreased in PFC and hippocampal tissues treated with GCs, amygdalar and PFC tissues treated with NVP, and PFC tissue treated with Ro-25. Overall, the results of the in vivo experiment did not convincingly demonstrate a role of glutamate spill-over in the pathogenesis of PTSD, but did show that modulation of glutamate reuptake can mitigate some of the behavioral consequences of exposure to situational trauma. The results of the ex vivo experiment did not reveal evidence that regional differences in NMDA receptor profiles exist across the three regions analyzed, nor did they show that GCs exert a region specific inhibition of long-term memory formation. However, it was demonstrated that SPS may affect long-term memory by altering expression of synaptic NMDA receptors. This study provides evidence that glial cells may play a role in the pathogenesis of PTSD, and thus may serve as targets for future therapy.
Evaluation of the toxic effects of eltenac (4-((2,6-dichlorophenyl) amino)-3-thiophene acetic acid), a nonsteriodal anti-inflammatory drug, in horses
Goodrich, Laurie Ruth (Virginia Tech, 1996-05-15)
A controlled study was performed to determine the potential toxic effects of the new nonsteroidal anti-inflammatory drug, eltenac (4-[(2,6-dichlorophenyl) amino]-3-thiopheneacetic acid), in horses. Four treatment groups were formed, each composed of 6 horses. The drug was injected intravenously, once daily, at a dose rate of 0.5 mg/kg, 1.5mg/kg or 2.5 mg/kg for 15 days. A control group was injected with sterile saline solution. Parameters assessed were hay and water consumption, daily clinical observations (evaluation of attitude, mentation, pulse and respiratory rate, fecal consistency, skin condition, and color and hydration of mucous membranes), physical examinations, complete hemogram, coagulation profiles, serum biochemical profiles, urinalysis, gastroscopic examinations and gross post-mortem and histopathological examinations on all organ systems. All examiners were blinded to group assignment and dosage levels until the completion of the study. A few glandular gastric ulcers, mild in severity, developed in seven animals during the treatment period. This occurred more often in horses treated with high doses of eltenac (P=.02). A dose dependent change of WBC count and neutrophil count were noted. Total protein, albumin and globulin levels had dose dependent decreases. One horse in the high dose group (2.5mg/kg) developed ventral edema as well as hypoproteinemia. N one of the horses in any of the dosage groups exhibited depression or anorexia. Gross post-mortem and histologic examination did not reveal any signs of drug related gastrointestinal, renal or hepatic abnormalities. Minimal toxic effects of eltenac given intravenously were greatest in horses treated with 2.5 mg/kg of the compound for 15 days.

Browsing by Author "Ehrich, Marion F."

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