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Browsing by Author "Lark, Daniel, committee member"

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    A novel approach to real-time monitoring of erythrocyte ATP release as a function of hypoxia
    (Colorado State University. Libraries, 2020) Smith, Meghan E., author; Dinenno, Frank, advisor; Lark, Daniel, committee member; Amberg, Gregory, committee member
    Background: Matching blood flow to tissue oxygen demand is essential for maintaining metabolic homeostasis and sustaining human life. Recent studies suggest that red blood cells (RBCs) play a role in local vasodilatory signaling by releasing ATP in response to hypoxia. RBC ATP release and overall function are impaired with age and disease. Until now, luciferin/luciferase bioluminescence is the only method described to quantify ATP release from RBCs. Here, we describe a novel approach where ATP release is measured as a function of hypoxia continuously and in real time using an Oroboros Oxygraph O2K respirometer. Purpose: This report describes the development and application of this new approach. Methods: We obtained blood samples from 10 healthy, young adults (18-35y) via venipuncture. Washed RBCs were diluted to 5% hematocrit and added to the glass chamber of a calibrated Oxyfluorimeter along with 5μM Mg-G. Nitrogen gas was constantly injected into the chamber at 1 ml/min to decrease PO2. An LED-based fluorescence detection device monitored Mg-G fluorescence, which was used to calculate extracellular [ATP]. Results: When 5% HCT RBCs were exposed to 30 minutes of hypoxia, Mg-G fluorescence (V) continuously increased. During 30 minutes of progressive hypoxia, PO2 in the chamber decreased from 121.9 ± 1.3 to 9.8 ± 0.8 mmHg, and D extracellular [ATP] from normoxia (μM) increases from 0 to 6,985.0 ± 793.6 μM. Extracellular [ATP] accumulates markedly when PO2 in the chamber reaches 50.60 ± 1.52 mmHg. Conclusion: Using this novel method, we identified a PO2 threshold at which extracellular ATP accumulates rapidly, which is consistent with the range of PO2 that elicits Hb desaturation in RBCs. This approach may allow for detailed mechanistic studies into the relationship between hypoxia, Hb desaturation, and RBC ATP release.
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    Mechanisms of exercise hyperemia during elevated oxygen delivery in humans
    (Colorado State University. Libraries, 2020) Anna, Jacob L., author; Dinenno, Frank, advisor; Richards, Jennifer, advisor; Lark, Daniel, committee member; Chicco, Adam, committee member
    The coupling between skeletal muscle oxygen delivery (O2D) and metabolic demand is largely attributed to the integration of feedback, and feedforward vascular mechanisms. It has been demonstrated that blood flow responses remain intact despite pharmacological elevations in resting blood flow, suggesting the existence of a vasodilator capable of augmenting hyperemia independent of tissue oxygen demand. We hypothesized that the change in forearm blood flow (FBF) from rest to steady-state exercise is preserved independent of baseline O2D, and that reciprocal reductions in oxygen extraction coincide with elevated O2D (Protocol 1). Additionally, pharmacological blockade of Kir channels and ATPase will reduce the change in FBF. In 10 young healthy adults, we quantified forearm blood flow (FBF; Doppler ultrasound), venous oxygen saturation (SvO2), oxygen extraction (O2 extraction; deep venous blood samples), and forearm oxygen consumption (mVO2) at rest and throughout 5 minutes of mild-intensity (10% maximal voluntary contraction; MVC) rhythmic handgrip exercise under control (CON) conditions and following intra-arterial infusion of the vasodilator sodium nitroprusside (SNP) to elevate local FBF and O2D. In Protocol 1, we elevated resting FBF and O2D to levels that matched (MAT) and exceeded (EXC) steady-state FBF (FBF: MAT; 166 ± 25 ml/min, P=NS, EXC; 219 ± 27 ml/min, P<0.05) during control (CON) exercise trials (FBF: CON; 172 ± 24). Changes in blood flow remained intact (ΔFBF: CON; 135 ± 20 ml/min vs. MAT; 132 ± 19 ml/min vs. EXC; 167 ± 26 ml/min, P=NS across all conditions), despite elevations in resting FBF which were adequate to sustain steady-state contractile activity under CON conditions. Reciprocal reductions in O2 extraction were observed in the MAT (O2 extraction: CON; 63 ± 3% vs. MAT; 43 ± 5%, P<0.05) and EXC trials (O2 extraction: CON; 63 ± 3% vs. EXC; 35 ± 5%., P<0.05) compared to CON during exercise. Additionally, we measured venous K+ in a subset of participants (N=6) to evaluate changes in K+ efflux (venous [K+] x FBF/1000) as an index of K+ release during exercise, alluding to K+-mediated activation of Kir channels and the ATPase. Five participants completed Protocol 2 which included control and elevated FBF trials (Saline + Block and SNP + Block) with the addition of intra-arterial infusion of barium chloride (BaCl2) and ouabain to inhibit Kir channels and the ATPase, respectively. Blockade of these pathways reduced the change in FBF that persisted during the Protocol 1 MAT trial (ΔFBF: MAT; 148 ± 21 ml/min vs. SNP + Block; 96 ± 13 ml/min, P<0.05). From this data, we are able to determine that changes in blood flow during exercise persist despite elevations in resting O2D (via SNP) prior to the start of exercise and that trends in O2 extraction follows changes in total O2D. We believe that local skeletal muscle K+ release is capable of activating Kir channels and the ATPase in a feed-forward manner which initiates a hyperpolarizing signal, thus augmenting blood flow independent of tissue oxygen demand.
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    Novel transcriptomic mechanisms of brain aging
    (Colorado State University. Libraries, 2023) Cavalier, Alyssa Nicole, author; LaRocca, Thomas, advisor; Lark, Daniel, committee member; Hamilton, Karyn, committee member; Weir, Tiffany, committee member
    As the world ages, the incidence of age-related diseases like dementia is expected to increase. Brain aging is characterized by declines in cognitive function that may develop into mild cognitive impairment, which increases the risk for dementia. In fact, age is the primary risk factor for late-onset Alzheimer's disease, which is the most common age-related dementia. The adverse cellular and molecular processes that underlie cognitive decline with aging in the brain are known collectively as the "hallmarks of brain aging." Advances in next-generation sequencing (e.g., transcriptomics/RNA-seq) have made it possible to investigate age- and disease-related changes in the brain at the broad gene expression level, and to identify potential therapeutic targets. With the support of my committee and mentoring team, I completed three studies using transcriptomics that characterize novel mechanisms that underlie brain aging. My findings include: (1) doxorubicin chemotherapy accelerates brain aging at the gene expression level, (2) apigenin nutraceutical supplementation targets age-related inflammation in the brain and rescues cognitive impairment in old mice, and (3) epigenetic dysregulation of transposable elements (remnants of viral infection in the genome) with aging contributes to age-related inflammation in Alzheimer's disease. Together, my work provides insight into transcripts and cellular/molecular pathways that are modifiable and may be therapeutic targets to delay or prevent consequences of brain aging.