Browsing by Author "Goodrich, Laurie R., committee member"
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Item Open Access Evaluation of kisspeptin in the mare(Colorado State University. Libraries, 2010) Magee, Christianne, author; Clay, Colin M., advisor; Tobet, Stuart A., committee member; Nett, Torrance M., committee member; Goodrich, Laurie R., committee member; Duval, Dawn L., committee memberIdentified in 2003 for their role in reproductive physiology, kisspeptins have become major players in the field of reproductive neuroendocrinology. With the ability to act as a central regulator for the onset of reproductive function in prepubertal and seasonal animals, the possibility that kisspeptin signaling could be used to modify seasonal reproductive function in the horse held great promise. My hypothesis was that kisspeptin, acting via a hypothalamic signaling mechanism to stimulate the GnRH neuron, could initiate reproductive function in the horse. The initial objectives of these studies were to (1) establish biological and physiological evidence for kisspeptin signaling in the hypothalamus of the mare, (2) demonstrate peripheral administration of kisspeptin could elicit a rise in serum luteinizing hormone (LH) concentrations in the diestrous mare, and (3) demonstrate that kisspeptin, acting via LH, could induce ovulation in the estrous mare. The diestrous mare has kisspeptin immunoreactive neurons in the hypothalamus that are in close proximity to Gonadotropin Releasing Hormone (GnRH) neurons. At the time of these initial studies, the equine sequence for the kisspeptin decapeptide (Kp-10) was not yet available; therefore, I utilized the rodent Kp-10 (rKp-10, YNWNSFGLRY-NH2). Even though I was using a heterologous ligand, the diestrous mare was responsive to administration of rKp-10 (0.5 and 1.0 mg) such that there was a short (< 1 hour), but significant (2-fold) rise in circulating levels of LH and follicle stimulating hormone (FSH) after kisspeptin administration. I was also able to establish a threshold dose for kisspeptin responsiveness in the diestrous mare as there was no change in serum gonadotropin levels following a 1.0 μg dose of rKp-10. In the estrous mare, a single injection of 1.0 mg rKp-10 IV was unable to induce ovulation (173), presumably due to the short duration of the kisspeptin induced LH surge as compared to the 3-5 day endogenous peri-ovulatory LH surge (306). To understand the dynamic of kisspeptin signaling to the hypothalamus and the anterior pituitary gland, I sought to determine the effect of treating mares with repeated injection of kisspeptide in diestrus and estrus. If the future of kisspeptin in the horse involves the use of modified agonists or antagonists, it will be necessary to understand how the mare responds to repeated stimulation with kisspeptin. Before beginning these studies, the equine sequence for Kp-I0 (eKp-10, YRWNSFGLRY-NH2) had become available. Therefore, I used the homologous peptide for these studies. By treating mares with eKp-10 (0.5 mg IV every 4 hours), the hypothalamus and pituitary gland were repeatedly stimulated to elicit a GnRH and gonadotropin response. Repeated administration of kisspeptin in the diestrous mare is not able to sustain a 2-fold increase in LH concentration for 48 hours following the initial injection. Interestingly, kisspeptin caused a decrease in basal LH, but not FSH levels, indicating a decrease in LH synthesis or secretion via a pituitary effect. Although the mare does not exhibit a change in peripheral LH levels following eKp-10 if a GnRH antagonist (e.g. Antide) has been administered, I sought some evidence for kisspeptin signaling directly to the anterior pituitary. To support the idea of a direct pituitary effect of kisspeptin, I challenged primary pituitary cells in culture with 100 nM GnRH and 100 nM of eKp-10. Surprisingly, I identified three populations of cells that respond with a change in intracellular calcium concentration and grouped them as follows: cells that responded to (1) both GnRH and eKp-l0, (2) only GnRH, or (3) only eKp-10. The identification of gonadotrope and non-gonadotrope kisspeptin responsive pituitary cells is the first evidence for a direct mechanism for kisspeptin signaling at the level of the equine pituitary gland. In the estrous mare, repeated administration of eKp-10 is not able to shorten the interval to ovulation whether it is administered before or after the development of a dominant follicle. Another surprising finding was a significant decrease in sexual receptivity in mares within 48 hours of beginning treatment with kisspeptin, which is likely due to a decrease in estradiol synthesis by the maturing follicle. Given the lack of ovulation induction in the estrous mare and the changes in behavioral receptivity, I do not recommend the use of kisspeptin as an ovulation inducing agent at this time. However, there was no decrease in basal LH levels in the estrous mares. Thus, kisspeptin may be signaling via different mechanisms in the estrous vs. diestrous mare. In summary, these studies do provide evidence for kisspeptin signaling in the mare, but they reveal that the signaling mechanism in the horse may be more complex than my original hypothesis of a simple, linear process that is working only through the GnRH neuron.Item Open Access From meniscus to bone: structure and function of human meniscal entheses and deleterious effects of osteoarthritis(Colorado State University. Libraries, 2013) Abraham, Adam Christopher, author; Haut Donahue, Tammy L., advisor; Kaufman, Kenton R., committee member; Puttlitz, Christian M., committee member; Popat, Ketul C., committee member; Goodrich, Laurie R., committee memberKnee osteoarthritis plagues millions of people in the U.S. alone, yet the mechanisms of initialization are not well understood. Recent work suggests that there are a myriad of potential disease inducing routes that may give rise to this debilitating condition. Understanding and elucidating the potential pathways leading to osteoarthritis may result in novel methods of prevention and/or treatment. Human meniscus are C-shaped fibrocartilaginous structures contained within the diathroidal knee joint, the primary function of which are to provide support and lubrication between the femur and the tibia. Each knee incorporates two menisci, lateral and medial, affixed at the anterior and posterior attachment sites to the tibial plateau. Meniscal attachments, or entheses, are unique graded tissue interfaces comprised of four distinct zones that diffuse longitudinal loads transmitted via hoop stresses of collagen fibrils in the meniscal body. The attachments must remain firmly rooted to the tibial plateau to effectively attenuate joint loads. If the attachments become structurally compromised, either through direct or indirect means, excessive transverse meniscal translation results. Such joint extrusion of the meniscal body is a known precursor to developing osteoarthritis. To date there have been no investigations of integrity of meniscal attachments in the aged arthritic knee. A proposed treatment modality for meniscus degeneration is engineered replacements which focus solely on the meniscal body, disregarding the specialized tissue interface. However, the efficacy of these replacements likely remains dependent on restoring the meniscus to bone transition. Previous literature has shown that each meniscal attachment is biochemically and mechanically unique and thus should be independently examined. Therefore, the overall goal of this work is to examine the loading environment of each attachment in both a healthy and injured knee, as well as characterize the structure-function relationship. This knowledge can then be utilized to develop novel preventative strategies in order to deter the onset of osteoarthritis, thereby reducing the burden on individuals as they age. Therefore, the goal of this work was to: • Determine the transverse mechanical properties of the attachment sites and couple with current literature to aid in numerical modeling • Determine the native loading environment for each attachment under physiological and pathlogical loading conditions • Examine the structure and function of the native attachment sites • Examine the deleterious effects of osteoarthritis on the attachment sites.Item Open Access Glial signaling mechanisms in the progression of neuroinflammatory injury(Colorado State University. Libraries, 2018) Popichak, Katriana A., author; Tjalkens, Ronald B., advisor; Bouma, Gerrit J., committee member; Goodrich, Laurie R., committee member; Legare, Marie E., committee member; McLean, Jennifer L., committee memberThe response of glial cells to foreign and endogenous stress signals is extensive. As a result, release of inflammatory factors as means of cellular communication and innate immune function, or neuroinflammation, can contribute to neurodegeneration and increased activation of surrounding glia, often associated with Parkinson's disease (PD). The identification of glial activation as an early event in the progression of neurodegenerative disease that precedes neuronal cell death presents an opportunity for better diagnostic markers, as well as new pathways that could be targeted therapeutically. The transcription factor, Nuclear Factor-kappa B (NF-κB), regulates the expression of multiple neuroinflammatory cytokines and chemokines in activated glial cells but the signaling factors modulating glial-glial and glial-neuronal signaling during neurotoxic injury are poorly understood. Thus, inhibition of NF-κB signaling in glial cells could be a promising therapeutic strategy for the prevention of neuroinflammatory injury. Recently, it was found that selected orphan nuclear receptors in the NR4A family (nerve growth factor-induced-β/NGFI-β), including NR4A1 (Nur77) and NR4A2 (Nurr1), can inhibit the inflammatory effects of NF-κB but there are no approved drugs that target these receptors. In the current studies, we utilized several experimental approaches to target neuroinflammation in cellular models of PD and manganese neurotoxicity in primary glia and in animal models. One of these studies demonstrated that a novel ligand of NR4A1 and NR4A2, 1,1-bis (3'-indolyl) -1-(p-methoxyphenyl) methane (C-DIM5), suppressed NF-κB-dependent inflammatory gene expression in astrocytes following treatment with 1-methyl-4-phenyl-1, 2, 3,6-tetrahydropyridine (MPTP) and the inflammatory cytokines, IFN-γ and TNF-α. These data were further supported by previous studies from our laboratory, which examined efficacy of multiple C-DIM compounds in PD animal and cellular models, including one (C-DIM12) identified as a modulator of Nurr1 activity that also inhibited NF-kB-dependent gene expression in glial cells. Collectively, these data demonstrate that NR4A1/Nur77 and NR4A2/Nurr1 dynamically regulated inflammatory gene expression in glia by modulating the transcriptional activity of NF-κB. An additional study examined the role of NF-κB in manganese (Mn)-induced neurotoxicity by exposing purified microglia, astrocytes (from both wild-type and an astrocyte- specific NF-kB (IKK2) knock-out (KO) mouse) and mixed glial cultures to varying Mn concentrations and then treated neurons with the conditioned media (GCM) of each cell type. In doing so, we showed that mixed glial cultures exposed to Mn enhanced glial activation and neuronal death compared to microglia, wild type astrocytes or IKK2-knockout astrocytes alone or in mixed cultures suggesting that astrocytes are a critical mediator of Mn neurotoxicity through enhanced expression of inflammatory cytokines and chemokines, including those most associated with reactive phenotype such as C3 and CCL2. Thus, these studies elucidate key mechanisms associated with neuroinflammation and present potential therapeutic targets in glial cells that regulate the progression of neuroinflammatory injury.Item Embargo Investigation into the mechanisms of bone loss in a sheep model of osteoporosis(Colorado State University. Libraries, 2024) Bisazza, Katherine T., author; Easley, Jeremiah T., advisor; Anthony, Russell V., committee member; McGilvray, Kirk, committee member; Goodrich, Laurie R., committee member; Nelson, Bradley B., committee memberOsteoporosis is the most common metabolic bone disease in humans and the leading cause of fragility fractures in the aging population. Given the invasiveness of researching bone diseases in people, appropriate animal models are essential to both build our understanding of the disease as well as examine novel therapeutics. While small animals and rodents are more commonly used as models in bone research, large animal models offer the ability to perform robust, long-term studies on bone quality with higher translational impact. Ovariectomized sheep are a well-established large animal model for osteoporosis because of the comparable bone size and microarchitecture that is shared with humans. While the ovariectomized sheep has been utilized for decades as a model for the study of bone, many gaps in the model characterization remain. Based on results from a preliminary literature search, we developed study objectives and hypotheses to expand upon current knowledge gaps in the characterization of the sheep model of osteoporosis. In order to test these aims, we performed a single 12-month in vivo study utilizing sixteen ewes. Osteoporosis was induced experimentally in ten of these ewes via ovariectomy followed by a 24-week regimen of high dose corticosteroids, while six ewes were used as healthy seasonal controls. In our first aim, we compared the bone density and microarchitectural changes in the osteoporotic animals as compared to healthy controls over the course of a year. Dual-energy x-ray absorptiometry (DXA) scans revealed significant bone density loss in the osteoporotic animals in both the lumbar spine and tibia as compared to control animals. We also noted significant microarchitectural changes in iliac crest bone biopsies of osteoporotic animals as indicated by micro-computed tomography (microCT), including decreased bone volume fraction, trabecular thickness, and trabecular number, as well as increased trabecular spacing. Additionally, we compared the use of quantitative computed tomography (QCT) and DXA to measure bone mineral density and correlated those findings with microarchitectural parameters in the osteoporotic animals. We demonstrated superior QCT sensitivity and specificity to subtle bone changes in the lumbar spine as compared to DXA, as well as demonstrated a higher correlation of QCT with iliac crest biopsy microarchitectural changes. The second aim of our study was to explore the systemic and clinical impacts of osteoporosis model development in our ten sheep compared to the healthy control animals. To test this aim, we collected blood, bone marrow, and body weights throughout the course of the year-long study. Osteoporotic animals demonstrated significant impacts to hematology and serology blood levels over the course of model development, primarily at 3 and 6-months when corticosteroids were at peak use. In particular, we note significant reductions in monocytes, lymphocytes, and eosinophils at 3-months with accompanying neutrophilia, as well as an increase in platelet count and volume. We also observed an increase in serum phosphorus and electrolytes, decrease in kidney enzymes and total protein, and an increase in select liver enzymes at 3 and 6-months in the same animals. Serum cortisol and estradiol were significantly depleted at 3 and 4-months, respectively, in the osteoporotic animals. However, estradiol levels were maintained to control levels for the remainder of the study. All these changes indicate disruptions to multiple physiologic systems over the course of osteoporosis induction in sheep which may highlight the acute effects of administering high-dose glucocorticoids. In the third and final aim of this study, we investigated the morphometrical and proteomic changes in the bone of sheep following osteoporosis induction over the course of a year. Histomorphometry of iliac crest bone biopsies revealed decreases in trabecular bone area in osteoporotic model animals compared to healthy controls, while negligible differences were observed in cortical bone morphometry. Initial global untargeted proteomic outputs identified a total of 4,765 proteins from the iliac bone biopsy samples, 909 of which were determined to be differentially expressed over the course of model development in our osteoporotic sheep. Pathway analysis of differentially expressed proteins (DEPs) revealed unique enriched pathways at all time points. Enrichment of biological processes such as monocyte differentiation, metabolic processes, regulation of chromosome condensation, and immune responses were noted throughout osteoporosis development. When comparing the 909 DEPs between time points, we identified seven downregulated proteins shared between all time points as compared to baseline in the osteoporotic animals (CTR9, INPP5D, CDK6, PPP2R5C, NUP133, ITPRIPL1, W5PH60_SHEEP). Pathway analysis of these shared proteins revealed enrichment of p53 signaling, mRNA surveillance, sphingolipid signaling, and P13K-Akt signaling pathways. This study was the first to report on the proteomic changes of bone in conjunction with morphometry assessments in a sheep model of osteoporosis. All three of our described experiments allowed us to successfully fill in some of the knowledge gaps in the characterization of a large animal model of osteoporosis by further assessing both macro and micro changes in ovariectomized and steroid-dosed sheep over the course of a year. Large animal preclinical models offer researchers the ability to compare bone changes in the same animals over time, allowing for a more comprehensive insight into the progression of postmenopausal and age-related bone loss. Understanding the mechanisms driving bone loss and systemic changes in osteoporosis disease progression could aid in future cellular therapy research and investigation of novel pathway targets for osteoporosis treatment in humans.Item Open Access Meniscal root tears and repairs(Colorado State University. Libraries, 2018) Steineman, Brett Daniel, author; Haut Donahue, Tammy L., advisor; LaPrade, Robert F., committee member; Goodrich, Laurie R., committee member; Heyliger, Paul R., committee memberMeniscal root tears are defined as radial tears of the meniscal insertions and lead to an inability for the menisci to transmit compressive loads into circumferential hoop stresses. These are common among the posterior meniscal insertions due to acute or chronic conditions. Anterior root tears have also been shown to occur from iatrogenic injury during anterior cruciate ligament reconstructions; however, the relationship between anterior insertions and the anterior cruciate ligament are understudied. Root tears of the posterior insertions lead to measurable osteoarthritis within a year if left untreated. Despite this, changes to tissue characteristics due to anterior root tears are unknown. If untreated anterior roots result in tissue degeneration, then it is important for both anterior and posterior root tears to be repaired to prevent, or at least delay, the onset of osteoarthritis. Meniscal root repair techniques have been developed to prevent joint degeneration following meniscal root tears; however, clinical studies of root repairs show that meniscal extrusion and joint degeneration are not completely prevented. This limited repair success may be due to inaccurate placement of repairs during surgery or from repair loosening postoperatively as early as during rehabilitation. The goals of this work are to better understand anterior root tears and to investigate potential causes for insufficient meniscal root repairs. Thus, the aims are to: 1) Quantify the overlap between the anterior cruciate ligament and the anterolateral meniscal insertion in the coronal and sagittal planes. 2) Assess early in vivo degeneration after untreated anterior meniscal root tears. 3) Determine the extent of repair loosening and recovery due to short-term rehabilitation. 4) Develop finite element knee models to determine the effect of repair placement and loosening on knee mechanics. The completion of this project will improve clinical practice and basic scientific knowledge of current issues facing meniscal root tears and repairs.