Browsing by Subject "Small or Companion Animal Medicine"
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Item Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties(1994-12-01) Tai-Lee, Ke; Clark, Abbot F.; Gracy, Robert W.; McConathy, Walter J.Ke, Tai-Lee, Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties. Doctor of Philosophy (Biochemistry), December, 1994, 139 pp., 26 tables, 13 illustrations, bibliography, 117 titles. This investigation was designed to characterize age-related changes in corneal function and biochemical structure. The specific aims were to: 1) systematically assess changes in permeability to compounds of different molecular weights and lipophilicities, 2) examine differences in tissue binding by utilizing a theoretical transport model, and 3) evaluate the biochemical changes in lipid composition and distribution. Experiments to compare young (six weeks) versus old (three to four years) rabbit corneal permeability were carried out utilizing an in vitro diffusion model. Changes in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. Permeabilities of selected compounds with different physicochemical properties were evaluated in young and old intact and denuded (wounded) rabbit corneas. With age, the membrane permeability significantly decreased in parallel with an increase in transmembrane resistance. Age-related changes in activities of esterase and phosphatase were also found. For some compounds, the aged corneas exhibited longer lag times in penetration studies. This suggested that the binding constant in the cornea from older animals was higher than in young animals. Maximum binding capacity from theoretical model calculations correlated well with experimental results in the young corneal stroma but correlation was less rigorous for old corneal stroma. Age-related changes in lipid composition and distribution in corneas were observed and provide indirect evidence for a decrease in membrane fluidity (decrease in the ratio of phosphatidylcholine/sphingomyelin) in the aged cornea. Results indicate that the aging process in the cornea is associated with changes in biochemical structural matrix including membrane lipid composition and physical properties such as fluidity (microviscosity). Functional correlations include changes in: 1) transmembrane resistance, 2) membrane permeability, 3) enzymatic activities (esterase and phosphatase), and 4) binding properties of the cornea. A possible mechanism for understanding and developing an intervention for age-related changes in the cornea is postulated.Item Delta Opiod Receptor: Parasympathetic Location and Changing Phenotypes in Canine Heart(2007-07-01) Deo, Shekhar H.; James L. Caffrey; H. Fred Downey; Michael SmithDeo, Shekhar H., Delta Opioid Receptor: Parasympathetic Location and Changing Phenotypes in Canine Heart. Doctor of Philosophy (Integrative Physiology), July 23, 112 pp, 4 tables, 24 figures. Delta opioid receptors (DOR) have long been implicated in the complex mechanism of ischemic preconditioning (IPC). Repeated arterial occlusion of the SA node artery in IPC protocol progressively raised the nodal encephalin concentrations and improved vagal transmission during a subsequent extended occlusion. This vagatonic effect was reversed by the DOR-1 antagonist, BNTX. The present thesis tested whether the IPC protocol, the prolonged occlusion or a combination of both was required to demonstrate the vagotonic effect. The study also tested whether the evolution of the vagotonic effect during occlusion might be attributed to erosion of completing vagolytic effects. A progressive improvement in vagal transmission was observed during the IPC protocol. The vagotonic effect was not observed during sham occlusions or during occlusions in animals pretreated with a DOR-1 antagonist. Following the IPC protocol, exogenous MEAP reduced vagal transmission under both normal and occluded conditions. The magnitude of the vagolytic effects was however significantly reduced and eroded further over time compared to time matched shams. The loss of the response was not altered by prior DOR-1. The magnitude of the vagolytic effects was however significantly reduced and eroded further over time compared to time matched shams, however the failure of DOR-1 blockade to slow that process suggests that the PC mediated erosion is independent of receptor activation by DOR-1 agonists. Although DORs are associated with IPC, their precise location remains unconfirmed. DOR and autonomic markers vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH) were labeled in tissue sections and synaptosomes from canine atrium and SA node. Synapsin I verified the neural character of labeled structures. Acetylcholine and norepinephrine content indicated both cholinergic and adrenergic synaptosomes are present. VAChT and TH signals indicated more than 80% of synapsin positive synaptosomes were cholinergic and less than 8% were adrenergic. Western blots of synaptosomal extracts confirmed by two DOR bands at molecular weights corresponding to reports for DOR monomers and dimmers. The preferential association of DORs with cholinergic nerve terminals supports the hypothesis that post-ganglionic prejunctional DORs regulate local vagal transmission within the heart.Item Differential Gene Expression Profiling in a Small Animal Model of Progressively Pacing-Induced Heart Failure(2006-06-01) Selby, Donald Evan; Stephen R. Grant; Patricia A. Gwirtz; Dan DimitrijevichDonald Evan Selby, Differential Gene Expression Profiling in a Small Animal Model of Progressively Pacing-Induced Heart Failure. Doctor of Philosophy (Biomedical Sciences), July 2006, 235 pp, 4 tables, 35 illustrations, references, 328 titles. Pacing induced tachycardia (PIT), in mammals, is known to cause a change from normal heart function to early left ventricular dysfunction. Progression to heart failure in experimental animals, such as dogs, pigs, and sheep, takes place in a relatively short period of time compared to the disease development observed in humans. Due to the cost and nature of using such animals, there is a need for a small animal model of PIT, which would delineate the etiology of the disease state by impairing the systolic function. The mode of action of overpacing inducement of cardiomyopathy, as the data suggests, may be through a sarcomere stretch sensor and its length-dependent signaling mechanism. In this study, an internal electrical-overpacing of an isogenic rabbit strain over a 52-day period was used to initiate a pathology consistent with human CHF. The data presented demonstrated that PIT causes alterations in the systolic ability of the heart, observed as reduced fractional shortening of the heart. This is seen in changes of the message pool population for proteins of the contractile architecture. Initially the heart is being paced rapidly and therefore there is insufficient time to get blood into the chamber. Thus, the data suggests that a mechanical stretch sensor is the process by which overpacing the heart leads to changes in gene expression which ultimately cause a compounding cellular condition which exists during heart failure. The data shows that there are gene isoform ratio changes that occur as the disease develops these include changes in differential expression of cardiac titin alternative splicing isoforms. The data suggests that there is also isoform switching occurring with alternative splicing of the gene encoding for SERCA2a, the probe 1587641_at shows a moderate decrease in expression and using BLAST for this probe this sequence is homologous to an alternative splicing variant of SERCA2a of the rabbit accession number J04703. The data shows that ferritin heavy chain also has an alternative splicing variant that are differentially regulated, this dysregulation of the isoform ratio may be linked to ADAMSTS1, a disintegrin and metalloproteinase isoform 1, which is seen to be downregulated in the data, these play a role in negative regulation of cellular proliferation. In addition to these detected isoform changes in the ratios of alternative splice variants changes are seen in genes linked to sarcomere integrity such as dystrophin probe 1582958_at is significantly increased in its expression, also integrin beta-1 probe 1584175)at shows a marginal increase in expression. The protease calpain probe 1604384)at, which uses a substrate the aforementioned integrin, dystrophin, and titin is also significantly upregulated in the data. Interestingly calpastatin, probe 1591603_at the inhibitor of calpain is marginally increased in its expression. Only recently has titin become to be appreciated as the protein that is responsible for the Frank Starling law as it undergoes an isoform ratio change as heart failure develops. These changes are initially caused by changes in ion concentration and stress upon the contractile proteins but as seen in the study, leads to altered gene expression. In this model, these gene alterations lead to diastolic dysfunction and the compounded problems constitute heart failure. This work shows that heart failure induced by over-pacing creates physical demands upon the framework of the heart and these physical stresses are transmitted through mechanical sensors leading to differential expression of the message pools for proteins involved in the way the heart contracts, and fills upon relaxation which ultimately ends in a heart that can do neither, thus leading to death.Item Dysfunctional Control of Coronary Blood Flow in Renovascular Hypertension(1999-06-01) Kline, Geoffrey Philip; Gwirtz, Patricia A.; Shi, Xiangrong; Raven, Peter B.Kline, Geoffrey Philip, Dysfunctional Control of Coronary Blood Flow in Renovascular Hypertension Doctor of Philosophy (Biomedical Sciences), June 1999, 98 pp, 2 tables, 10 figures, references, 142 titles. This study was designed to determine the effects of renovascular hypertension (RVH) on coronary vasoreactivity in conscious, chronically instrumented dogs. Six dogs were instrumented to measure left ventricular pressure, +dP/dtmax, heart rate, mean aortic pressure, circumflex blood flow (CBF), and cardiac output. In order to examine endothelial-dependent and independent coronary vasodilation, intracoronary injections of actylcholine (Ach), bradykinin (BDK), and sodium nitroprusside (SNP) were studied before and after induction of RVH in the presence and absence of nitric oxide (NO) blockade. After RVH, resting CBF was significantly reduced (P [less than] 0.05). In the normotensive state, NO-blockade significantly reduced the coronary vasodilation to Ach and BDK (P [less than] 0.05), but not SNP. After RVH, the coronary vasodilation to Ach, BDK, and SNP were reduced (P[less than] 0.05). After RVH, NO-blockade further reduced the coronary vasodilation to BDK (P [less than] 0.05), but not Ach. Thus, RVH resulted in an impairment of both endothelial-dependent and –independent coronary vasodilation. It also appears that during RVH the endothelium retains the ability to produce/release NO to some, but not all, stimuli. In order to examine the possibility that β-adrenergic mediated coronary vasodilation is impaired after RVH, intracoronary injections of norepinephrine (NE), isoproterenol (ISO), and terbutaline (TRB) were administered. These drugs all caused dose dependent increases in CBF before and after RVH. After RVH, the coronary vasodilatory responses to NE, ISO and TRB were significantly reduced (P [less than] 0.05). β1-blockade with intracoronary atenolol (1 mg) reduced the ISO-induced increases in CBF and had no effect on TRB responses (P [less than] 0.05). β2-blockade with intracoronary ICI-118,551 (1 mg) reduced the ISO-induced coronary vasodilation and abolished TRB responses (p[less than] 0.05). During β2-blockade, ISO-induced increases in CBF were not different after RVH. Therefore, these data indicate that β1-adrenergic mediated coronary vasodilation is preserved after RVH, whereas, β2-mediated is not. We conclude that 1) RVH results in an impairment of both endothelial-dependent and –independent coronary vasodilation; 2) RVH results in an impairment of β2-adrenergic mediated coronary vasodilation.Item Functional Heterogeneity in Canine Coronary Resistance Arteries(1994-06-01) Parker, James Bruce; Peter B. Raven; Patricia A. Gwirtz; James CaffreyParker, James B., Functional Heterogeneity in Canine Coronary Resistance Arteries. Doctor of Philosophy (Biomedical Sciences), June, 1994, 89 pages, 21 illustrations, bibliography, 82 titles. Two thirds of the coronary vascular resistance resides in the smallest arteries and investigators have hypothesized that they may respond differently to endogenous vasoactive substances. The arterial responses to norepinephrine, acetylcholine, and adenosine were evaluated in large ([greater than] 700 μm, n=24), intermediate (400 600 μm, n=24), and small arteries (μm, n=24). Maximal vessel lumen diameter (Dmax) was determined in CA++ free medium. A reference diameter (84 ± 4.3% of Dmax) was established by re-equilibration in medium containing 2.0 mM Ca++. Arterial maximal responses as a percentage of Dmax to norepinephrine, acetylcholine, and adenosine are given in table 1: Table 1; Large % of Dmax; Inter. % of Dmax; Small % of Dmax; Norepinephrine; 41 ± 2.3; 50 ± 4.2; 83 ± 2.4; Acetylcholine; 96 ± 2.7; 88 ± 3.9; 78 ± 1.9; Adenosine; 71 ± 1.8; 81 ± 4.2; 96 ± 1.4. The sensitivity of canine coronary arteries to norepinephrine, acetylcholine, and adenosine in terms of ED50’s are given in table 2: Table 2; Agonists; Large ED50 μM; Inter. ED50 μM; Small ED50 μM; Norepinephrine; 0.037 ± 0.002; 0.078 ± 0.004; no response; acetylcholine; 0.028 ± 0.003; 0.087 ± 0.005; 0.309 ± 0.03; Adenosine; 0.295 ± 0.002; 0.095 ± 0.004; 0.035 ± 0.03. These data indicate that canine arterial responses to the native agonists norepinephrine, acetylcholine, and adenosine are heterogeneous and that neural control predominates in the larger “transport” arteries while local control predominates in the smaller “distributive” arteries. Responses of small and intermediate isolated canine coronary arteries (lumen diameter 147±42μm, and 531±37μm respectively) to norepinephrine were evaluated after pharmacological or mechanical interruption of endothelial relaxing activity. Following with the nitric oxide synthase inhibitor N-Nitro-L-Arginine Methylester (L-NAME) 10^-5 M the small and intermediate vessels spontaneously constricted to 73±4.1% of Dmax indicating a significant basal release of nitric oxide. After L-NAME or endothelial disruption graded additions of norepinephrine now reduced the vessel diameter in previously unresponsive small arteries. These data suggest that the weak and equivocal response of coronary resistance arteries to norepinephrine results from the competitive dilatory influence of endothelial derived nitric oxide production and not to the absence of norepinephrine receptors.Item Mechanisms of Right Ventricular Oxygen Supply/Demand Balance in the Concious Dog(2000-06-01) Hart, Bradley; H. Fred Downey; Patricia A. Gwirtz; James L. CaffreyHart, Bradley Joe. Mechanisms of Right Ventricular Oxygen Supply/Demand Balance in the Conscious Dog Doctor of Philosophy (Biomedical Sciences), August,2000, 119 pp, 4 tables, 13 figures, references, 79 titles. No data exist in the literature describing the myocardial oxygen supply/demand relationship of the right ventricle in a conscious, anaesthetized animal. A novel technique developed in our laboratory enables us to collect right ventricular (RV) venous blood samples from conscious dogs to determine RV myocardial oxygen consumption (MVO2). RV oxygen supply/demand balance was examined in conscious dogs, chronically instrumented to measure right coronary blood flow (RCBF), segmental shortening (%SS) and RV pressure (RVP) during increases and decreases in RV myocardial oxygen demand. Right ventricular MVO2 and O2 extraction (O2E2) were determined; RCBF, RVP, dP/dt, and %SS were recorded concomitantly. Acute increases in RV MVO2 were accomplished by atrial pacing (200 beats/min), increasing RV afterload by 65%, infusion of isoproterenol (0.1 μg/kg/min, i.v.), and by conducting a submaximal exercise routine (70-75% of maximum VO2). An acute decrease in RV MVO2 was created by propranolol administration (1 mg bolus, i.c.). During acute increases in RV MVO2, the extraction reserve is utilized primarily; flow is not affected in the absence of direct vasodilatory effects of the intervention. A decrease in RV oxygen demand is associated with a further increase in the RV extraction reserve. Since RV O2E increases linearly with increases in RV MVO2, these data show that changes in RV venous O2 tension can occur with little or no change in RCBF. LC resistance is very sensitive to alterations in LC venous pO2; therefore, there appear to be significant differences between the left and right ventricles concerning the matching of oxygen supply with myocardial oxygen demand.Item Regulation of Myocardial Blood Flow and Function During Exercise in Dogs(1995-06-01) Kim, Song-Jung; Patricia A. Gwirtz; Peter B. Raven; James L. CaffreyIntroduction. Background. Coronary circulation during exercise. Coronary blood flow is regulated primarily by local metabolic mechanisms according to the oxygen and nutrient needs of the heart (2, 4, 19). The local “metabolic signal” involves vasoactive metabolites, such as adenosine, released from myocytes in direct proportion to myocardial work (Figure 1). However, other external factors are superimposed on local regulatory mechanisms and can substantially modulate coronary blood flow. One of these modulatory factors is the sympathetic nervous system. Sympathetic vasoconstriction mediated by α-adrenergic receptors in the coronary circulation has been shown to oppose metabolic vasodilation and limit oxygen supply to the myocardium during physiologic and pathophysiological cardiac stresses, such as exercise and myocardial hypoperfusion (1, 6, 7, 8, 10-14, 17, 18, 21). This limitation on myocardial oxygenation appears to impose a restriction on the increase in regional left ventricular subendocardial contractile function during submaximal exercise (7). In this regard, studies have shown that removing this α1-constrictor tone leads to an increase in coronary blood flow and, as a result, regional contractile function (8). This adrenergic coronary constriction during exercise is mediated by neutrally released norepinephrine, not by circulating catecholamines (8). Endothelial-mediated control of coronary vascular tone. Recent investigations indicate that another factor involved in modulating coronary blood flow is the vascular endothelium. The endothelium exerts an influence on vascular smooth muscle vasomotor tone by releasing an endothelium-derived relaxing factor (EDRF) or nitric oxide (NO), which is derived from the amino acid L-arginine by nitric oxide synthase (5, 22). Synthesized NO diffuses into the underlying vascular smooth muscle to activate cytosolic guanylate cyclase (GC), thereby stimulating the intracellular accumulation of cyclic GMP (cGMP). This is illustrated in Figure 2. NO is released by the stimulation of muscarinic receptors on endothelial cells by acetylcholine, as well as by other agonists or physical stimuli (e.g., shear stress) at the interface between blood and endothelial cell surface (15). During exercise, for example, the work output of the normal heart may increase several-fold by the stimulation of sympathetic nerves to heart. The increased work output of the heart increases myocardial oxygen demand. Consequently, the coronary circulation undergoes vasodilation due to local metabolic mechanisms. The elevation in shear stress caused by increases in coronary blood flow triggers release of NO from the endothelium because of the extremely pulsatile nature of the flow. Therefore, it is likely that during exercise, release of NO by shear stress and by neurohormonal stimuli, concomitant with local release of metabolites, contributes to coronary dilation. These vasodilatory influences counteract a sympathetic α-adrenergic coronary constriction, which limits the increase in coronary blood flow and cardiac performance. Accordingly, coronary vascular smooth muscle tone during exercise is modulated by the endothelium, which responds to the increased shear stress and adrenergic stimulation, which provides the major extrinsic input.Item Role of Adenosine in Acute Hibernation of Guinea-Pig Myocardium(1995-08-01) Gao, Zhi-Ping; H. Fred Downey; James L. Caffrey; Patricia A. GwirtzGao, Zhi-Ping, Role of Adenosine in Acute Hibernation of Guinea-Pig Myocardium Doctor of Philosophy (Biomedical Sciences), August, 1995; 111 pp; 3 tables; 15 figures, bibliography, 158 titles. Myocardial hibernation is a state of depressed contractile function and energy demand during chronic ischemia. When coronary flow is restored, depressed contractile function can partially or completely recover to the pre-ischemic level, and ischemic injury of the myocardium in not evident. This project tested the hypothesis that endogenous adenosine mediates hibernation in guinea-pig myocardium. Isolated working guinea-pig hearts, perfused with glucose fortified Krebs-Henseleit buffer, were subjected to global low-flow ischemia. Left ventricular performance and cytosolic energy level were assessed. Lactate and purine nucleotides were measured in venous effluent. Heart were perfused with [U-14C]glucose to investigate the role of adenosine on glucose metabolism in myocardium. Left ventricular function in untreated hearts decreased by 80% and remained stable during ischemia, and completely recovered upon reperfusion. Neither adenosine receptor blockade with 8-p-sulfophenyl theophylline (8-SPT; 20 μM) nor ecto 5’-nucleotidase inhibitor αβ-methylene adenosine 5’-diphosphonate (AOPCP; 50μM) affected left ventricular function either ischemia or during reperfusion. Cytosolic energy level fell by 67% at 10 min ischemia in untreated hearts, but subsequently recovered to the pre-ischemic level despite continued ischemia. Adenosine receptor blockade increased cytosolic energy level at 10 min ischemia relative to untreated hearts, but blunted the subsequent rebound of phosphorylation potential. Moreover, 8-SPT doubled ischemic lactate release. Adenosine receptor blockade also increased glucose uptake during pre-ischemia and hypoperfusion, but did not stimulate glucose oxidation. Crossover plots of glycolytic intermediates revealed that phosphofructokinase, a key rate-controlling step in glycolysis, was activated by adenosine receptor blockade in both pre-ischemic and hibernating myocardium. We conclude that 1) activation of adenosine receptors results in recovery of cytosolic energy level of moderately ischemic working myocardium, but this energetic recover is not solely responsible for post-ischemic contractile recovery; 2) endogenous adenosine attenuates anaerobic glycolysis during myocardial hibernation by blunting phosphofructokinase activity.