Browsing by Author "Tobet, Stuart, advisor"
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Item Open Access Design and evaluation of an instrumented microfluidic organotypic device and sensor module for organ-on-a-chip applications(Colorado State University. Libraries, 2020) Richardson, Alec Evan, author; Henry, Charles, advisor; Tobet, Stuart, advisor; Bark, David, committee member; Abdo, Zaid, committee memberOrgan and tissue-on-a-chip technologies are powerful tools for drug discovery and disease modeling, yet many of these systems rely heavily on in vitro cell culture to create reductionist models of tissues and organs. Therefore, Organ-on-chip devices recapitulate some tissue functions and are useful for high-throughput screening but fail to capture the richness of cellular interactions of tissues in vivo because they lack the cellular diversity and complex architecture of native tissue. This thesis describes the design and testing of 1) a microfluidic organotypic device (MOD) for culture of murine intestinal tissue and 2) a microfluidic sensor module to be implemented inline with the MOD for real-time sensing of analytes and metabolites. The MOD houses full-thickness murine intestinal tissue, including muscular, neural, immune, and epithelial components. We used the MOD system to maintain murine intestinal explants for 72 h ex vivo. Explants cultured in the MOD formed a barrier between independent fluidic channels perfused with media, which is critical to recapitulating intestinal barrier function in vivo. We also established differential oxygen concentrations in the fluidic channels and showed that more bacteria were present on the tissue's mucosal surface when exposed to near-anoxic media. The sensor module is a reversibly sealed microfluidic device with magnetic connections that can withstand high backpressures. Further, electrodes housed in commercial finger-tight fittings were integrated into the sensor module in a plug-and-play format. Future work will include developing electrochemical/optical sensors for various biological compounds relevant to intestinal physiology. Ultimately, the MOD and sensor module will be implemented in long-term microbiome studies to elucidate the relationship among microbial, epithelial, neuro and immune components of the gut wall in health and disease.Item Open Access Estradiol exposure alters gonadothropin-releasing hormone (GNRH) induced gonadotrope plasticity(Colorado State University. Libraries, 2010) Hartshorn, Cheryl, author; Tobet, Stuart, advisor; Clay, Colin, committee member; Hentges, Shane, committee member; Tjalkens, Ron, committee memberThe reproductive axis is dependent upon communication among the hypothalamus, pituitary and gonads. For successful ovulation, a large increase in circulating estradiol provides positive feedback at both the hypothalamic and pituitary levels to promote an luteinizing hormone (LH) surge. An LH surge is necessary for the final maturation of the pre-ovulatory follicle and ovulation. The cellular and molecular events underlying estradiol’s action(s) upon the anterior pituitary gland, specifically gonadotropes, remain elusive. Recent video microscopy experiments showed that pituitary cells in vitro in slice culture move in response to GnRH [Navratil, et al., 2007]; presumably these cells were gonadotropes. The current study utilized a novel transgenic animal model that has gonadotrope specific fluorescence provided by yellow fluorescent protein (YFP) [Wen et al., 2008]. I sought to determine if 17(3-estradiol (E2) working through either a genomic or non-genomic mechanism affected gonadotrope specific movements in response to GnRH. Consistent with earlier studies [Navratil et al., 2007], application of GnRH [100nM] altered the cytoarchitecture of gonadotropes with observable cell process extensions. Using live video- microscopy, exposure to 10nM E2 for fourteen hours significantly enhanced the ability of gonadotropes to extend processes in response to GnRH compared to short-term exposure of E2 (1.5 hours) or vehicle. There was no demonstrable effect of 1.5 hours of E2 exposure on GnRH-induced process extensions. I hypothesize that the differential effect of short-term versus long-term E2 exposure is due to a genomic mechanism that may underlie the ability of E2 to enhance GnRH induced cellular plasticity. Thus, E2 and GnRH may cooperate to maximize the secretory interface between gonadotropes and the adjacent vasculature during the pre-ovulatory LH surge.Item Open Access Gamma-aminobutyric acid (GABA) in the development of the paraventricular nucleus of the hypothalamus (PVN): implications for adult disease(Colorado State University. Libraries, 2012) Stratton, Matthew S., author; Tobet, Stuart, advisor; Amberg, Gregory, committee member; Bamburg, James, committee member; Mykles, Donald, committee memberThe paraventricular nucleus of the hypothalamus (PVN) is the final common regulator of the neuroendocrine stress response. Humans with depression or anxiety disorders display altered regulation of this system and females are more likely to suffer from these disorders than males. This work investigated embryonic development of the PVN to identify cellular processes that might occur incorrectly (preferentially in females) and predispose the individual to altered regulation of stress responses. GABA acts as a neurotrophic factor during development. As the embryonic PVN is ringed by GABA (absence of GABA in the PVN) and a receptor for GABA is enriched in the PVN, it was hypothesized that this molecule would direct PVN development. Embryonic development was altered in mice either by genetic manipulation (receptor knockout mice) or by pharmacological blockade of the GABAA and GABAB receptors. Embryonic GABAA receptor antagonism caused a decrease in the number of neurons that expressed estrogen receptor α in and around the PVN. In female but not male mice lacking GABAB receptors, the cytoarchitecture of the PVN was altered. Specifically, estrogen receptor containing cells were misplaced and corticotropin releasing hormone was increased. Animals treated with a GABAB receptor antagonist during embryonic development copied the phenotype of receptor knockout mice. The in vivo effect of GABA signaling on cell placement was investigated in vitro with organotypic slice fluorescence video microscopy. Again only in females, blockade of the GABAB receptor caused neurons to increase migration speed. Thus GABA acts to restrict cells from moving outside of the PVN. When the GABAB receptor is antagonized, cells migrate outside of the PVN. To determine the consequence of an animal having altered PVN development, animals treated as embryos with the GABAB receptor antagonist were subjected to a battery of behavior tests as adults. Interestingly, females treated embryonically with CGP 55845 displayed an increased anxiety-like phenotype (female specific disorder) while males treated with the same compound displayed a hyperactivity-like phenotype (male specific disorder). Independent of sex, animals treated as embryos with the GABAB receptor antagonist displayed decreased depression-like behaviors and had a less robust stress response compared to vehicle treated animals. This work highlights the importance of GABA signaling in PVN development and the dependence of complex adult behaviors on embryonic brain organization as GABA receptor antagonism limited to a specific critical time period during embryonic development recreated cytoarchitectural and behavioral phenotypes of GABA receptor knockout mice.Item Open Access Glucocorticoid-dependent regulation of molecular clocks and dendritic spines in the ventromedial prefrontal cortex(Colorado State University. Libraries, 2022) Miller, Alex M., author; Tobet, Stuart, advisor; Vigh, Jozsef, committee member; Bamburg, Jim, committee member; Maresh, Ryan, committee memberBiological rhythms in the brain and periphery are governed by the suprachiasmatic nucleus of the hypothalamus (SCN) and the SCN's control of rhythmic adrenal glucocorticoid (GC) secretions via the hypothalamic pituitary adrenal (HPA) axis. The daily surge of GC secretions aid in the entrainment of molecular clocks throughout the body which physically, mentally, and metabolically prime an organism to function in accordance with the external light:dark (L:D) cycle. When key events of biological rhythms fail to match up with the external L:D cycle, various pathologies arise in the brain and periphery. To better understand the neural basis of pathologies caused by the disruption of biological rhythms, further investigation of key limbic regulatory brain regions is required. Thus, the studies described in this dissertation examine how biological rhythms in the ventromedial prefrontal cortex (vmPFC) are regulated by GC secretions. The vmPFC regulates fear acquisition, fear extinction, mood, and HPA axis function. Multiple brain regions exhibit time-of-day dependent variations in learning, long term potentiation (LTP), and dendritic morphology. GCs have been implicated in the regulation of dendritic structure in the context of stress. GCs are also known to regulate molecular clock entrainment via upregulation of Per1 transcription in a variety of tissues. In the present study, C57BL/6N mice were sacrificed at 3 distinct times of day (ZT3, ZT12, and ZT16, lights off at ZT12) and Per1 mRNA expression was measured in the infralimbic and prelimbic vmPFC subregions using droplet digital (dd)PCR after recovering from adrenalectomy or sham surgery for 10 days. Sham mice showed Per1 rhythmicity in both IL and PL, with peak expression occurring at ZT12. Adrenalectomized mice showed reductions in Per1 amplitude at ZT12 in both IL and PL, suggesting that the vmPFC molecular clock is entrained by diurnal GC oscillations. Thy1-eGFP mice were used to visualize and quantify dendritic spine density on layer V pyramidal dendrites at ZT 3, 12, and 16. Spine density in both PL and IL exhibited changes between the light (inactive) and dark (active) phases, with peak spine density observed at ZT16 and trough spine density observed at ZT3. These changes in spine density were restricted to changes in long thin and stubby type spines. To determine if changes in spine density is regulated by diurnal GC oscillations, the 11β-hydroxylase inhibitor metyrapone was administered 2 hours prior to the onset of the active phase (ZT10) daily for 7 days. Metyrapone administration blocked both the diurnal peak of plasma corticosterone and peak spine densities in the IL and PL at ZT16. These results suggest that vmPFC molecular clock gene and dendritic spine diurnal rhythms depend on intact diurnal GC oscillations. These findings establish a link between diurnal GC oscillations, the molecular clock, and synaptic plasticity. Additionally, these findings describe how the vmPFC changes across 24-hour periods, which provides a foundation for further investigation into how biological rhythms in the vmPFC may be altered in the context of circadian disruption, and how specific disease states may arise as a result.Item Open Access Sex dependent regulation of immune responses in ex vivo lung slices(Colorado State University. Libraries, 2023) Patlin, Brielle Honor, author; Snow, Chris, advisor; Tobet, Stuart, advisor; Chanda, Soham, committee memberSex differences in respiratory disease have been increasingly obvious over the last several decades ranging from asthma, to interstitial lung disease, to the common cold. One way that lung functions are dependent on sex is in their immune responses to disease. While there are many factors that contribute to the severity of immune responses and recovery from illness, neuroimmune signaling is an understudied aspect. This could be partially caused by the difficulty of studying individual neuronal circuits in the periphery in live animals and the lack of necessary cell types in organoid or cell line models. To study these processes in the lung, an organotypic model, known as a precision cut lung slice (PCLS) can be utilized to maintain intracellular and extracellular signaling. The first study herein addresses the role of the most prevalently produced neuropeptide in the lung on immune, neuronal, and epithelial populations in the lung. This abundant neuropeptide, calcitonin gene related peptide (CGRP), generates several sex dependent responses in PCLS. With CGRP treatment, number of B cells, size of neuroendocrine bodies (NEB), and surfactant protein C (SPC) granule are higher in female PCLS. However, the number of CGRP immunoreactive fibers in female PCLS is lower than in male PCLS. These sex related changes of lung cell behavior may partially explain some disease susceptibilities and are important factors to consider in pharmaceutical development for respiratory diseases. PCLS can be used to test pharmaceutically relevant substances and drug delivery systems. The recent pandemic has made it evident that better ways to deliver pharmaceuticals to the lungs are required. Regarding this problem, a focus on nasal drug delivery is important. The current leading technology in this type of delivery mechanism involves utilizing lipid nanoparticles and nasal administration. However, this is not the most efficient way to treat the lungs. Crystallized protein structures have begun to be used for purposes other than determining protein structure. In the second study included here, protein crystals were loaded with biologically relevant molecules to purposefully induce immune responses, without causing an immune response by themselves. This functionality has a variety of benefits, because a primary problem in respiratory disease is over activation of the immune response. In this study, crystals were customized by loading different molecules (e.g., lipopolysaccharide (LPS)) and the immune modulatory affects were observable in PCLS. This generated a sex dependent immune response in PCLS, which was less over time than slices treated with pure LPS indicating a differential response over 48 hours. Using protein nanocrystals for pharmaceuticals may provide new ways to target respiratory disease by nasal delivery with benefits over lipid nanoparticles.Item Open Access Stress during pregnancy leads to long-term consequences in the offspring(Colorado State University. Libraries, 2024) Sheng, Julietta Angelina, author; Tobet, Stuart, advisor; Smith, Bret, advisor; Myers, Brent, committee member; Hale, Taben, committee member; Chanda, Soham, committee memberNeuropsychiatric disorders encompass a wide range of conditions that affect neurological health and brain function and lead to disabilities worldwide. Such disorders include, but are not limited to, Major Depressive Disorder, schizophrenia, and anxiety disorders. Risk factors for developing neuropsychiatric disorders are multifaceted and can range from genetic predisposition, lifestyle, and environmental influences. Exposure to maternal stress is one type of environmental factor that can lead to changes in brain function and signaling pathways and increase susceptibility for related diseases. Maternal stress encompasses a diverse array of environmental stimuli, ranging from acute traumatic events to chronic or day-to-day life stressors. Maternal stressors, experienced by pregnant women, lead to overexposure of stress hormones in the developing fetus and impact short- and long-term neurological health the offspring. These studies evaluated developmental, neuroendocrine, and behavioral outcomes in offspring exposed to different models of maternal stress. Chapter 1 provided a brief history of stress, the development of the hypothalamic-pituitary-adrenal axis that regulates the stress response, and maternal-fetal interactions in stress regulatory systems and related behaviors. Chapter 2 evaluated several models of maternal stress, maternal high fat diet, maternal caloric restriction, maternal exposure to synthetic glucocorticoids. Although there were vast discrepancies between each type of maternal stress, one similarity was an activated immune response with elevated maternal cytokines. Therefore, Chapter 3 characterized a model of maternal immune activation using a toll-like receptor agonist, Resiquimod, that increased maternal and fetal cytokines, produced delayed developmental milestones and stress-related behavioral impairments in prepubertal (social-like) and adult (social-like, depressive-like, anxiety-like) offspring. Because these behavioral phenotypes are partially regulated by the paraventricular nucleus of the hypothalamus (PVN), Chapter 4 examined the neuroendocrine stress response and blood-brain barrier of the PVN. Data showed altered stress response accompanied by impaired blood-brain barrier integrity in the PVN of the adult offspring exposed to maternal injection of Resiquimod. Taken together, Chapters 2, 3, and 4 suggest maternal stress led to negative developmental, behavioral, and cellular pathologies indicative of neuropsychiatric-like disease. By teasing apart these specific programming mechanisms, we can better diagnose and treat progression of neuro-related disorders.Item Open Access Two types of melanopsin retinal ganglion cell in the mouse retina: the regulation of melanopsin expression(Colorado State University. Libraries, 2009) Baver, Scott Benjamin, author; Tobet, Stuart, advisorRods, cones and a subset of retinal ganglion cells (RGCs) that express the photopigment melanopsin are the sensory photoreceptors of the mammalian retina. The light-driven signals that are initiated by the photoreceptors are relayed from the retina to the brain. In addition to the role of light information in regulating the perception of colors, objects and movement, it also controls pupil size and the synchronization of daily physiological rhythms to the day/night cycle. The melanopsin-expressing RGCs, which are intrinsically photosensitive (ipRGCs), contribute especially to these two latter processes. The focus of this dissertation is the ipRGCs of the mouse retina.Item Open Access Using a precision-cut lung slice co-culture paradigm to increase T-cell populations and model infection ex vivo(Colorado State University. Libraries, 2024) Ehrlich, Alexis T., author; Tobet, Stuart, advisor; Perera, Rushika, committee member; Magee, Christianne, committee memberPrecision cut lung slices (PCLS) bridge a gap between in vivo and in vitro studies by maintaining anatomical organization with structural integrity and intercellular signaling pathways. Applications of PCLS have included the modeling of inflammatory lung diseases, metabolism studies, and drug development. In the lungs, immune responses are carried out by a network of T- and B- cells, the latter of which are resident. The limited resident T-cell population of the lung diminishes accurate representations of pathogen response capacity in PCLS. Addressing this, we set out to increase pulmonary T-cell populations ex vivo. We hypothesized that thymus and bone marrow-derived T-cells would work synergistically to populate the lung in co-culture experiments. A murine organotypic lung co-culture model was developed and characterized for tissue health and T-cell recruitment over 3 days ex vivo using adult neurobasal media with 4 mM glucose + 2% B27 supplement. Lung slices were cultured independently, with bone marrow, thymus, or both. Immune colonization of the lung was assessed using immunohistochemistry for CD3+ T-cells, CD19+ B-cells and ACK2+ cells. Cells were counted in alveolar and airway spaces after 3 days of culture. Our results demonstrate that lung co-cultured with thymus and bone significantly increased T-cell populations ex vivo, whereas lung co-cultured with thymus or bone alone did not significantly alter T-cell counts. Additionally, B-cells and C-Kit+ cell populations were not influenced by the culture paradigm. Using this paradigm, we went on to explore this lung co-culture paradigm when stimulated by an immune modulatory agent – LPS- and when an active lung infection is present using Pseudomonas aeruginosa. Lung and lung co-cultures had increases in T-cell counts after immune stimulation and infection. Additionally, the co-cultures further increased T-cell counts after treatment. More strikingly, the co-cultures influenced the degree of bacterial infection in the lung slices without altering the B-cell populations among cultures. Viral infections are also common pathogens that affect the lungs, so we examined then examined the ability of viral pathogens to infect precision cut lung slices. As we did not get an infection with live virus, we explored the effect of a viral mimic – Resiquimod- on lung co-cultures on the immune responses. Resiquimod and the co-cultures are both able to increase T-cell populations in lung slices ex vivo. These results suggest that the increased T-cell population corresponding with thymus and bone marrow co-culture could be a result of cell-cell interaction or the secretion of growth factors. Cell secretions or growth factor release could stimulate thymic secretion of T-cells or could stimulate T-cell proliferation in the lung, suggesting that co-culture with thymus and bone marrow can elicit a T-cell response ex vivo. T-cells are necessary for host-pathogen immune responses, most commonly by CD8+ T-cells but there are other populations of T-cells. Although there are limitations to the use of lung slices in infection studies, the results of the co-culture and increasing T-cells is a promising step to studying pathogen response capacity in the future ex vivo.