Browsing by Author "Santangelo, Kelly, advisor"

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Open Access
Cleavage of exosomal-associated transferrin receptor in dogs, cats, and horses: progress towards a soluble transferrin receptor assay
(Colorado State University. Libraries, 2018) Martinez, Caitlyn Marisa Romero, author; Olver, Christine, advisor; Santangelo, Kelly, advisor; Duval, Dawn, committee member
Iron deficiency anemia and anemia of chronic disease are two complications that patients in human medicine as well as veterinary medicine often encounter. These two diseases usually occur secondary to other primary diseases and are associated with increased morbidity and a decline in prognosis. The diagnosis and differentiation of these diseases is complicated by the fact that many of the parameters used to characterize iron deficiency are also influenced by inflammatory cytokines. Consequently, the detection of iron deficiency in the presence of inflammation or the detection of combined iron deficiency anemia and anemia of chronic disease is difficult. Differentiation of these two diseases is important as treatment for each disease is different and potentially harmful if utilized on a patient who has been misdiagnosed with one disease or the other. In human medicine, soluble transferrin receptor 1 (sTfR) has shown promise as a marker, alone or in ratio with serum ferritin, that can differentiate iron deficiency anemia, anemia of chronic disease, and combined disease. sTfR is the product of cleavage of transferrin receptor 1 (TfR1) from the surface of exosomes which are released into circulation from maturing reticulocytes. Humans cleave the majority of their exosomal-associated TfR1 yielding substantial levels of circulating sTfR for detection and quantification by clinical assays. However, the level of cleavage in many of our veterinary species, including dogs, cats, and horses, remains unknown. Additionally, no currently developed sTfR clinical assays have been found to successfully detect sTfR in our veterinary species. The purpose of this study was to first confirm the presence of exosomes and exosomal-associated TfR1 in the serum of dogs, cats, and horses. Secondly, the level of cleavage of exosomal-associated TfR1 in healthy dogs, cats, and horses was explored to indirectly characterize the anticipated levels of circulating sTfR in these species. Lastly, the level of cleavage of exosomal-associated TfR1 was compared between healthy and diseased dogs and cats to investigate any potential effect of inflammation and chronic disease on the cleavage of exosomal-associated TfR1 and thus on the anticipated levels of circulating sTfR. The results of this study demonstrated significant evidence indicating the successful isolation of exosomes and identification of exosomal-associated TfR1 from the serum of dogs, cats, and horses. The level of cleavage of exosomal-associated TfR1 in dogs was found to be greater than 50% on average with significant between-individual variation. There was also no significant difference in the means of the proportion of cleavage between healthy and diseased dogs. The level of cleavage of exosomal-associated TfR1 in cats was found to be very low at about 11% without substantial variation between individuals. However, a small but significant difference between healthy and diseased cats was detected. Healthy horses do not appear to cleave exosomal-associated TfR1. These results together would suggest that development of a clinical assay for the detection and quantification of sTfR in these veterinary species may not be successful and consequently may not be worth the time, effort, and expense.
Open Access
Elucidating the role of iron in the pathogenesis of idiopathic osteoarthritis in the Dunkin-Hartley animal model
(Colorado State University. Libraries, 2021) Burton, Lindsey Hammond, author; Tjalkens, Ronald, advisor; Santangelo, Kelly, advisor; Legare, Marie, committee member; Goodrich, Laurie, committee member; Argueso, Lucas, committee member
Osteoarthritis (OA) is the most prevalent musculoskeletal disorder, affecting millions of individuals worldwide. While OA is characterized by the progressive loss of articular cartilage, it is now widely accepted to be a whole joint disorder, with changes such as synovial hyperplasia, subchondral bone remodeling, and osteophyte formation accompanying cartilage degeneration. The knee is one of the joints most affected by OA. Patients with knee OA exhibit painful and/or limited mobility as a consequence of the disorder, resulting in an increased risk of comorbidities such as heart disease, obesity, diabetes, and depression. Unfortunately, the mechanisms driving OA pathogenesis remain poorly understood, and there are no effective therapies available for treating the disorder. Therefore, there is a need to understand factors contributing to OA to identify potential targets for combating the condition. Iron is the most abundant mineral in the human body and is essential for conducting numerous physiologic processes. However, unbound or partially-liganded iron can participate in redox reactions that produce reactive oxygen species and free radicals capable of inciting tissue damage. As such, iron needs to be tightly managed within the body. Mammals do not possess a regulated mechanism for excreting iron, and iron progressively accumulates within tissues throughout the aging process. Primary/idiopathic OA does not have any known, identifiable cause for disease development, but the largest risk factor associated with the disorder is advancing age. To address this gap in knowledge, we designed a series of experiments to elucidate the contributions of iron to the pathogenesis of idiopathic OA. The main animal model used for this work is the Dunkin-Hartley guinea pig, which spontaneously develops age-related OA with a histopathology similar to that observed in humans. In the first study, we quantified tissue iron levels at different ages in OA-prone Dunkin-Hartley guinea pigs relative to an outbred, control strain not used in OA research. While the control strain accumulated iron in the liver with age, but not within cartilage, the Hartleys demonstrated a significant increase in cartilage iron concentration at 7-8 months-of-age. This increase in cartilage iron concentration was more significant in males, though it was also observed in females. As this timepoint corresponds to a moderate stage of disease progression, this finding suggests that iron may play a role in OA development and/or progression in Hartley guinea pigs. This concept was supported by gene expression analysis of iron-related genes. Notably, both male and female Dunkin-Hartley guinea pigs had decreased transcript expression of ferritin heavy chain and ferroportin at 7-8 months, which may contribute to cartilage iron accumulation at this age by inappropriately storing iron in chondrocytes. Because of this intriguing association, we wanted to investigate the gene expression changes occurring with systemic iron manipulation in knee joint tissues. Exogenous iron overload resulted in worsening of OA pathology in the disease-resistant Strain 13 guinea pig. The systemic administration of iron dextran caused iron to accumulate within articular cartilage from a diarthrodial joint environment and was accompanied by gene expression changes within knee tissues. Notably, systemic iron overload altered the expression of several iron-related genes in this control strain, indicating that both the cartilage and a large adipose depot, the infrapatellar fat pad, were able to detect and respond to changes in tissue iron levels in the presence of joint pathology. Conversely, systemic iron deficiency, achieved by supplying an iron deficient diet, decreased cartilage lesions within OA-prone male Hartley guinea pigs. In this proof-of-principle study, the reduction in cartilage iron concentration was accompanied by the altered expression of two iron transport genes, the importer transferrin receptor 1 and the cellular iron exporter ferroportin. As iron deficiency is not a recommended pursuit, we investigated the effects of systemic iron reduction, without clinical iron deficiency or anemia, on OA pathogenesis. The commercially available pharmacologic iron chelator deferoxamine (DFO) was used to reduce total iron levels in the body of male and female Dunkin-Hartley guinea pigs. In males, administration of DFO was successful at reducing tissue iron levels both systemically and in a diarthrodial joint environment, and this was accompanied by a significant decrease in the severity of cartilage lesions. The reduction in joint pathology observed with treatment was largely attributed to a decrease in chondrocyte cell death; this finding was supported by the decreased expression of several proapoptotic genes within knee articular cartilage. Conversely, tissue iron levels were not altered by administration of the same dose of DFO in females, suggesting the presence of sex differences in systemic iron homeostasis. There was a relative reduction in histologic OA score in treated female animals, which may be due to the beneficial mobilization of iron by DFO that was also noted in males. The modest reduction in female joint pathology with treatment was largely driven by decreased tidemark advancement. Tidemark replication is associated with articular cartilage mineralization and was almost completely absent in all males evaluated, implying there may also be differences in OA pathogenesis between male and female Dunkin-Hartley guinea pigs.
Open Access
Evaluation of acupuncture as an alternative therapy in a rodent model of spontaneous osteoarthritis
(Colorado State University. Libraries, 2020) Personett, Alexa R., author; Santangelo, Kelly, advisor; Kendall, Lon, committee member; Hamilton, Karyn, committee member
Osteoarthritis (OA) is a degenerative joint disease affecting nearly 250 million people globally. With clinical signs of severe and persistent joint pain, OA is a leading cause of physical disability throughout the world. When faced with the frustration of chronic discomfort and restricted mobility due to OA, many individuals have turned to acupuncture as an alternative therapy. Acupuncture is a traditional Chinese practice of medicine for pain alleviation that involves insertion of thin needles into the skin and underlying tissue. The needles may be manipulated via manual or electrical stimulation, referred to as manual acupuncture and electroacupuncture, respectively. However, the efficacy of acupuncture in managing OA pain is uncertain, as much of the evidence is of questionable quality. The overall goal of this project was to evaluate acupuncture in a rodent model of human OA such that unbiased conclusions regarding its effectiveness for symptom modification could be drawn. Unfortunately, the majority of laboratory models of OA are artificially induced via chemicals or surgery and may not adequately represent the spontaneous disease process that occurs in humans. In contrast, the Dunkin Hartley guinea pig is a natural disease model, with primary OA pathology that mirrors human disease. As the major symptoms of OA are painful and decreased mobility, we were interested in evaluating the effect of acupuncture on a variety of mobility parameters using treadmill-based gait analysis and open-field enclosure monitoring. Additionally, as OA is an inflammatory disorder, we were interested in evaluating the effect of acupuncture on systemic inflammation, as well as any potential effects on normal physiology. This is commonly done in veterinary species with minimally invasive blood tests, such as complete blood counts and serum biochemistries. Despite their widespread use in biomedical research, there are few published studies investigating normal reference ranges for these diagnostics in the Dunkin Hartley guinea pig. Therefore, the goal of the first study was to develop hematology and serum biochemistry reference intervals for this strain. Data from complete blood counts and serum biochemistries were compiled from control Dunkin Hartley guinea pigs from previous studies to establish reference ranges for this model. Data were stratified by sex to determine specific reference intervals for males and females, and significant differences in parameters were investigated based on age and sex. The results of this study provide a foundation for interpreting these common diagnostic and laboratory blood tests in the Dunkin Hartley strain. The second and third studies evaluated electroacupuncture and manual acupuncture for the treatment of OA utilizing the Dunkin Hartley guinea pig model. Results of these studies demonstrated that electroacupuncture improved mobility, but not structural changes, in the knee joint. Conversely, manual acupuncture did not improve mobility parameters, but trended toward a decrease in knee joint histology scores compared to control groups. The results of these studies provide evidence that short-term treatment with electroacupuncture, but not manual acupuncture, is effective for symptom modification in this animal model of OA. Long-term studies are still needed to determine mechanisms for disease modification with these modalities.
Open Access
Evidence for acute inflammation in the pathogenesis of different mouse models of post-traumatic osteoarthritis
(Colorado State University. Libraries, 2022) Timkovich, Ariel E., author; Santangelo, Kelly, advisor; Olver, Christine, committee member; Moreno, Julie, committee member; Goodrich, Laurie, committee member
Post-traumatic osteoarthritis (PTOA) is a debilitating, degenerative condition affecting more than 5.6 million people in the United States. Following injury to the knee, a person is 4.2 times more likely to develop PTOA in that joint, compared to an uninjured joint. PTOA is characterized by clinical symptoms such as pain, swelling, and decreased range of motion of the knee or affected joint. As a leading cause of pain and disability, PTOA is a major contributor to a decreased quality of life. Notably, loss of mobility associated with PTOA can increase the risk of a multitude of other ailments including heart disease and diabetes. PTOA due to traumatic joint injury can result in damage to intra-articular structures, such as menisci, ligaments, cartilage, and subchondral bone. Despite current treatment options, many of these joint injuries progress to end-stage PTOA within 10 to 20 years. Rupture of the most commonly injured knee ligament, the anterior cruciate ligament (ACL), typically occurs in athletes and people who are physically active, with the majority of ACL tears occurring in patients who are less than 30 years old. The detrimental pathways driving PTOA remain loosely defined and little progress has been made in predicting, preventing, and treating the disease. The goal of this work was to further our understanding of PTOA mouse models through both development and characterization of models, as well as through the testing of therapeutics in pre-established PTOA mouse models. In the first part of this work (Chapter 2), we created and characterized a novel full and partial ACL rupture model. This work is the first published partial ACL rupture model to date and provides a novel platform to use as a preclinical model for testing of potential therapeutics and to further our understanding of PTOA following ACL rupture. Chapter 3 evaluates the therapeutic potential of a toll-like receptor 4 (TLR4) antagonist on a full depth PTOA model. We found that short term treatment with a systemic TLR4 antagonist significantly improved relevant gait parameters, and improved cartilage structural metrics and modified Mankin PTOA scores, implying improvement of clinical signs. Chapter 4 addresses the therapeutic potential of intra-articular bone marrow aspirate concentrate (BMAC), for treatment of PTOA in a well characterized meniscus degeneration model, compared to red blood cell (RBC) depleted BMAC or no injection controls. These results suggest that RBC-depleted BMAC improved treatment of PTOA in an animal model and may have translational therapeutic potential for humans suffering from similar disease processes. Finally, Chapter 5 sought to: (1) Identify transcriptional sex differences in knee joint tissue from naïve animals; and (2) Identify compensatory gene expression changes related to sex that are specific to animals receiving DMM surgery as compared to a naïve animal when taking into account surgery changes (i.e., sham surgery). The work described in this dissertation has improved our understanding of the mouse knee joint during PTOA progression and potential therapeutics and targets for clinical treatment of PTOA.
Open Access
Optimization of overhead enclosure monitoring software in a rodent model of osteoarthritis
(Colorado State University. Libraries, 2022) Helbling, Joel E., author; Santangelo, Kelly, advisor; Easley, Jeremiah, committee member; Kendall, Lonnie, committee member
Osteoarthritis (OA) is a degenerative joint disease characterized by pain, inflammation, and decreased range of motion, leading to impaired activities of daily living and reduced quality of life. OA affects between 250 and 500 million people worldwide, contributing to a substantial and sustained economic burden. Given the global pervasiveness of this poorly understood disease process, in vivo OA research relies on both naturally occurring and induced animal models for its study. The Dunkin Hartley guinea pig spontaneously develops degenerative joint disease as early as 3 months of age and represents a well-characterized animal model of primary OA with pathological progression similar to humans. In contrast, secondary OA is caused by non-idiopathic factors, including trauma, and animal models of secondary OA rely on chemical, surgical and non-surgical induction of instability. Open-field testing (OFT) is a behavioral tool which provides objective measurements of mobility outcomes for animals enrolled in musculoskeletal studies and can be paired with overhead monitoring software to non-invasively track voluntary animal movement through the designated arena. However, established protocols for OFT have not been published in the guinea pig. The overarching goal of this project was to optimize OFT in the guinea pig to reduce environmental variability in behavioral testing conditions. The results of this project provided a framework to ensure accurate and reproducible data collection in subsequent studies involving therapeutic interventions to both spontaneous OA and traumatic OA. A hallmark symptom of OA is pain and, as such, the second portion of this work was dedicated to researching cannabidiol (CBD) as an alternative interventional therapeutic to analgesia. Specifically, mobility outcomes assessments were performed during a pharmacokinetic safety study as well as a chronic oral CBD dosing study. Significant differences were analyzed both on baseline (pre-treatment) and on treatment intervention in each phase of this two-part study pertaining to OFT. The results of these studies identified time-of-day effects exist when testing guinea pigs in the open-field and provided preliminary evidence that no adverse short-term behavioral effects exist after oral administration of CBD. The final goal of this project was to design of bioreactor to establish a non-surgical animal model of post-traumatic osteoarthritis (PTOA) in the guinea pig through precision rupture of the anterior cruciate ligament (ACL) by tibial compression and displacement. While this model has been characterized in other rodents, it has not been described in guinea pigs. Work from this portion of the project helped produce a functional bioreactor which will be used initially on cadavers and will ultimately promote in vivo research of interventional treatments for PTOA by establishing reproducible ligament lesions with subsequent degenerative joint pathology.
Open Access
The roles of iron, the infrapatellar fat pad, and dietary factors in the Hartley guinea pig model of spontaneous osteoarthritis
(Colorado State University. Libraries, 2018) Radakovich, Lauren, author; Santangelo, Kelly, advisor; Olver, Christine, committee member; MacNeill, Amy, committee member; Foster, Michelle, committee member; Pagliassotti, Michael, committee member
Osteoarthritis (OA) is the most prevalent musculoskeletal disorder across the world, affecting close to 300 million people. The disease manifests as degeneration and loss of articular cartilage, synovial hyperplasia, formation of osteophytes, subchondral bone remodeling, and joint space narrowing. These changes result in decreased range of motion as well as painful mobility in affected individuals. The knee joint is the most commonly afflicted joint. Osteoarthritic changes may develop secondary to a localized injury, referred to as post-traumatic OA. Degenerative changes can also develop without an inciting cause, which is referred to as spontaneous, or primary OA. Spontaneous OA is an insidious disease that is associated with aging, and, more recently, with obesity. The mechanisms contributing to disease development are not yet fully characterized, which has impeded implementation of successful treatment options. Currently, there are no treatments that are able to restore degraded cartilage. Thus, most patients with symptomatic knee OA undergo costly total knee joint replacement surgeries. The aims of this dissertation were to explore the roles that aging-associated iron accumulation, the infrapatellar fat pad, and calorie restriction with various diets may play in OA development. These studies were performed in the Hartley guinea pig, one of the only small animal models of spontaneous OA. One study was performed in Strain 13 guinea pigs, a strain that is OA-resistant. Iron is an element that acts as a double-edged sword. It is essential for oxidative phosphorylation and heme synthesis, yet its redox potential means it has the capacity to incite oxidant damage when present in excess. As there is no direct excretion mechanism for iron, it tends to accumulate slowly within tissues over time. This cellular iron accrual has been implicated in many degenerative diseases associated with aging, but its potential role in spontaneous OA has not been well-studied. In our first studies, we demonstrated that systemic iron levels have an influence on OA. We were able to induce bony and cartilage lesions in OA-resistant Strain 13 guinea pigs by systemic administration of iron dextran. Immunohistochemistry (IHC) indicated higher levels of lipid peroxidation in cartilage, menisci, the infrapatellar fat pad (IFP), and synovium in the iron overload animals. Special stains revealed that iron content was significantly higher in the IFP in these animals, which we propose serves as a local depot of oxidant damage to the knee joint. In a parallel study, we fed OA-prone Hartley guinea pigs an iron deficient diet to determine if reducing systemic iron levels may have a protective effect on the knee joint. Cartilage lesions were significantly lower in the iron deficient diet group compared to controls. Likewise, IHC for lipid peroxidation revealed less oxidant damage in the iron deficient pigs. However, no differences were noted in knee joint iron content, so the exact mechanisms for the lessened OA remain unclear. Because the iron overload study pointed to the IFP as a potential iron depot, we wanted to further characterize how this adipose tissue contributes to overall knee joint homeostasis. First, we demonstrated that quantitative iron content in the IFP was increased in aged, osteoarthritic guinea pigs compared to young, healthy animals. Gene expression data collected suggested that dysregulated iron trafficking, particularly increased expression of ZIP14 – which has been linked to pathologic iron uptake in other conditions – may be contributing to this aging-associated increase of iron in the IFP. Because of our suspicion that the IFP may be inciting local oxidant damage to the knee, we surgically removed it from a set of young Hartley guinea pigs. An identical sham procedure was performed in the contralateral limb. Four months post-surgery, animals were collected to evaluate OA in both limbs. Both cartilage and bony OA scores were markedly decreased in the IFP removal limb compared to the sham surgery limb. It is possible that removal of the IFP removed a source of local inflammatory mediators and iron, which resulted in lessened OA. As the IFP was replaced by a thick band of fibrous connective tissue, increased joint stability was also considered a contributing factor. Future studies will more closely examined contributions of biomechanical factors that may be at play. Finally, we aimed to determine how dietary manipulations may influence early OA, as previous studies suggest that calorie restriction may improve end-stage OA. Additionally, many studies have shown high fat diet (HFD)-induced obesity plays a role in OA development due to the inflammatory nature of excess adipose tissue. In our study, we demonstrated that calorie restriction with a low fat regular chow diet, but not a calorie restricted HFD, delayed onset of OA in Hartley guinea pigs. In fact, the HFD group had higher levels of systemic inflammation than the restricted regular chow group. The HFD group had similar levels of inflammation and OA scores as obese animals. Thus, we concluded that the pro-inflammatory nature of a HFD supersedes any positive effects of calorie restriction in the onset of spontaneous OA.