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Nonuniform cardiac denervation observed by 11C-meta-hydroxyephedrine PET in 6-OHDA-treated monkeys. | |
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PMID: 22539969 Owner: NLM Status: MEDLINE |
Parkinson's disease presents nonmotor complications such as autonomic dysfunction that do not respond to traditional anti-parkinsonian therapies. The lack of established preclinical monkey models of Parkinson's disease with cardiac dysfunction hampers development and testing of new treatments to alleviate or prevent this feature. This study aimed to assess the feasibility of developing a model of cardiac dysautonomia in nonhuman primates and preclinical evaluations tools. Five rhesus monkeys received intravenous injections of 6-hydroxydopamine (total dose: 50 mg/kg). The animals were evaluated before and after with a battery of tests, including positron emission tomography with the norepinephrine analog (11)C-meta-hydroxyephedrine. Imaging 1 week after neurotoxin treatment revealed nearly complete loss of specific radioligand uptake. Partial progressive recovery of cardiac uptake found between 1 and 10 weeks remained stable between 10 and 14 weeks. In all five animals, examination of the pattern of uptake (using Logan plot analysis to create distribution volume maps) revealed a persistent region-specific significant loss in the inferior wall of the left ventricle at 10 (P<0.001) and 14 weeks (P<0.01) relative to the anterior wall. Blood levels of dopamine, norepinephrine (P<0.05), epinephrine, and 3,4-dihydroxyphenylacetic acid (P<0.01) were notably decreased after 6-hydroxydopamine at all time points. These results demonstrate that systemic injection of 6-hydroxydopamine in nonhuman primates creates a nonuniform but reproducible pattern of cardiac denervation as well as a persistent loss of circulating catecholamines, supporting the use of this method to further develop a monkey model of cardiac dysautonomia. |
Valerie Joers; Kailie Seneczko; Nichole C Goecks; Timothy J Kamp; Timothy A Hacker; Kevin G Brunner; Jonathan W Engle; Todd E Barnhart; R Jerome Nickles; James E Holden; Marina E Emborg |
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Publication Detail:
Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2012-04-23 |
Journal Detail:
Title: PloS one Volume: 7 ISSN: 1932-6203 ISO Abbreviation: PLoS ONE Publication Date: 2012 |
Date Detail:
Created Date: 2012-04-27 Completed Date: 2012-09-10 Revised Date: 2014-05-07 |
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Nlm Unique ID: 101285081 Medline TA: PLoS One Country: United States |
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Languages: eng Pagination: e35371 Citation Subset: IM |
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MeSH Terms | |
3,4-Dihydroxyphenylacetic Acid
blood Animals Blood Pressure / drug effects Carbon Radioisotopes / chemistry Dopamine / blood Echocardiography Ephedrine / analogs & derivatives*, blood, chemistry, diagnostic use Female Heart / radionuclide imaging* Macaca mulatta Male Neurons / drug effects Norepinephrine / blood Oxidopamine / chemistry, toxicity* Positron-Emission Tomography Primary Dysautonomias / diagnosis Radiopharmaceuticals / chemistry, diagnostic use* |
Grant Support | |
Chemical | |
Reg. No./Substance:
0/Carbon Radioisotopes; 0/Radiopharmaceuticals; 102-32-9/3,4-Dihydroxyphenylacetic Acid; 3002-40-2/3-hydroxyephedrine; 8HW4YBZ748/Oxidopamine; GN83C131XS/Ephedrine; VTD58H1Z2X/Dopamine; X4W3ENH1CV/Norepinephrine |
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Journal Information Journal ID (nlm-ta): PLoS One Journal ID (iso-abbrev): PLoS ONE Journal ID (publisher-id): plos Journal ID (pmc): plosone ISSN: 1932-6203 Publisher: Public Library of Science, San Francisco, USA |
Article Information Download PDF Joers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received Day: 3 Month: 11 Year: 2011 Accepted Day: 16 Month: 3 Year: 2012 collection publication date: Year: 2012 Electronic publication date: Day: 23 Month: 4 Year: 2012 Volume: 7 Issue: 4 E-location ID: e35371 ID: 3335153 PubMed Id: 22539969 Publisher Id: PONE-D-11-22193 DOI: 10.1371/journal.pone.0035371 |
Nonuniform Cardiac Denervation Observed by 11C-meta-Hydroxyephedrine PET in 6-OHDA-Treated Monkeys Alternate Title:Nonuniform Cardiac Denervation in Monkeys | |
Valerie Joers12 | |
Kailie Seneczko1 | |
Nichole C. Goecks1 | |
Timothy J. Kamp3 | |
Timothy A. Hacker3 | |
Kevin G. Brunner1 | |
Jonathan W. Engle4 | |
Todd E. Barnhart4 | |
R. Jerome Nickles4 | |
James E. Holden14 | |
Marina E. Emborg124* | |
Thomas Hummeledit1 |
Role: Editor |
1Preclinical Parkinson's Research Program, Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America |
2Neuroscience Training Program, University of Wisconsin-Madison, Madison, Wisconsin, United States of America |
3Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America |
4Department of Medical Physics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America |
Technical University of Dresden Medical School, Germany |
Correspondence: * E-mail: email@example.com Contributed by footnote: Conceived and designed the experiments: ME JH TK. Performed the experiments: VJ KS NG TH TK KB JH JE TB RJN ME. Analyzed the data: VJ JH TH TK ME. Contributed reagents/materials/analysis tools: JH ME TK TH. Wrote the paper: VJ JH ME. |
Parkinson's disease (PD) is a movement disorder associated with degeneration of the nigrostriatal dopaminergic pathway; however, patients also experience nonmotor symptoms, with up to 80% having some form of autonomic dysfunction [1], [2], [3], [4]. Dysautonomias greatly affect PD patients' quality of life, and often are more disabling than motor deficits [5].
Cardiac dysautonomia, or abnormal autonomic control of the heart, is characterized in PD by orthostatic hypotension, an increase in corrected QT intervals (QTc), and reductions in heart rate variability and plasma norepinephrine [6], [7], [8], [9], [10]. Cardiac denervation, which may be a component of dysautonomias or found independently in PD patients, [11], [12], [13], [14] is associated with arrhythmias, shortness of breath during exercise, reduced time to peak heart rate, and fatigue [6], [15]. High co-prevalence suggests cardiac denervation and dysautonomia are intimately related with serious clinical consequences. New screening tools [7] and treatments are needed.
Cardiac sympathetic loss in PD has been documented using sympathoneuronal imaging agents such as 6-[18F]fluorodopamine [11], [16], [17], [18] and 11C-meta-hydroxyephedrine (MHED) [19], [20] with positron emission tomography (PET) and [123I]metaiodobenzylguanidine with single-photon emission computed tomography [12], [13], [21], [22], [23]. Low uptake in the left ventricle (LV) myocardium is reported in all stages of PD, although specific patterns of loss are not well described. Both diffuse and localized losses to lateral and inferior LV walls are reported [12], [20], [24]. Regardless of the pattern, loss is progressive [6]. PD patients exhibited as much as 30% loss of 6-[18F]fluorodopamine uptake in the LV lateral wall over 2 years, with relative preservation in the septal wall [18]. Loss of cardiac sympathetic innervation is confirmed post mortem by nearly complete absence of tyrosine hydroxylase, an enzyme in catecholamine biosynthesis, and by reduced neurofilament protein, a marker for the presence of axons [25], [26], [27].
Development of PD treatments targeting cardiac dysautonomia requires comprehensive animal models mimicking this symptom, [28] but no well-characterized nonhuman primate model of cardiac dysautonomia is currently available. Systemic administration of 6-hydroxydopamine (6-OHDA) to mice [29], [30] and dogs [31], [32] induces persistent loss of cardiac catecholaminergic innervation, supporting use of 6-OHDA to model cardiac dysautonomias in PD. We therefore aimed to assess the feasibility of developing a monkey model of cardiac dysautonomia by administering 6-OHDA within a system of in vivo evaluation in order to generate preclinical tools toward understanding this condition and testing therapies for PD patients.
The present study was performed in strict accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals (7th edition, 1996) in an AAALAC accredited facility (Wisconsin National Primate Research Center, University of Wisconsin - Madison). Experimental procedures were approved by the Institutional Animal Care and Use Committee of the University at the Wisconsin-Madison (permit number: G00538). All efforts were made to minimize the number of animals used and to ameliorate any distress.
Five adult rhesus monkeys (Macaca mulatta; 5–9 years old; 6–10 kg; two male, three female) were used in this project. The study followed a within-subjects experimental design (Figure 1) in which each animal was used as its own control. This design facilitates matching subjects between groups, reduces the chance of confounding factors, and the number of monkeys used. The animals were individually housed in Group 3 or Group 4 enclosures (cage floor area 4.3 ft.2 or 6.0 ft.2 per animal, height 30 or 32 in.) in accordance with the Animal Welfare Act and its regulations and the 7th edition of the Guide for the Care and Use of Laboratory Animals (1996) with a 12-hour light/dark cycle. Throughout the study, the animals were monitored twice daily by an animal research technician or veterinary technician for evidence of disease or injury (e.g., inappetance, dehydration, diarrhea, depression, lethargy, trauma, etc.) and body weight was documented weekly to ensure animals remained in properly sized cages. Animals were fed commercial nonhuman primate chow (2050 Teklad Global 20% Protein Primate Diet, Harlan Laboratories, Madison, WI) twice daily, supplemented with fruits or vegetables and a variety of forage items and received ad libitum water. Nonhuman primate chow soaked in a protein-enriched drink (Ensure©, Abbott Laboratories, Abbott Park, IL) was offered to stimulate appetite as needed after neurotoxin dosing.
6-OHDA hydrobromide (Sigma-Aldrich, St. Louis, MO) solution was prepared under a certified chemical hood, less than 2 hours before administration. The solution was kept out of light and on ice until immediately before administration, when it was drawn up into a 10-mL syringe covered with foil, and flushed through an amber-colored infusion line. Animals were food-deprived overnight; anesthesia was induced with ketamine HCl (15 mg/kg im) and maintained with 1–3% isoflurane in 100% O2 at 1 L/min. 6-OHDA (total final dose 50 mg/kg) was mixed into a sterile ascorbic acid solution (1 mg/mL 0.9% NaCl) and administered intravenously in a series of 5 mL injections (see Table 1 for dosing scheme) at a rate of 1 mL/min, using a motorized syringe pump (KD Scientific, Holliston, MA). Dosing was based on previous reports administering 6-OHDA to dogs [31], [32]. Blood pressure, heart rate, respiration rate, blood oxygen, and electrocardiograms (ECG) were monitored throughout the procedure; their normalization (return to baseline values) defined timing of subsequent dosing. Before dosing, blood was taken for complete blood chemistry and hematocrit measurements; additional hematocrit measurements were performed after each dosing.
Heart rate, cardiac auscultation, complete blood count, and blood chemistry were performed at least monthly before and after 6-OHDA. Feces were monitored daily by trained personnel and their characteristics recorded using a descriptive scale. The feces were identified as: no stool, very little stool, firm stool, soft feces (feces not-formed, usually soft or loose), diarrhea (watery or fluid feces), and mucus feces.
A trained observer monitored for PD signs using a previously validated clinical rating scale [33], [34]. The scale rates tremor (0–3 for each arm), posture (0–2), gait (0–5), bradykinesia (0–5), balance (0–2), gross motor skills (0–4 for each arm), defense reaction (0–2) and freezing (0–2). The total score ranges from 0 (normal condition) to 32 points (extreme severe disability).
A 10-lead ECG (Hewlett-Packard PageWriter, MA) was performed at baseline, during toxin administration, and at 1, 4, 10, and 14 weeks after 6-OHDA. Data were collected at least 15 minutes after starting isoflurane anesthesia. Electrodes were placed on identical positions on the interior right and left arms and legs, the 4th intercostal space immediately right of the sternum (V3), the 5th intercostal space immediately left of the sternum, and directly across from V3 at the anterior axillary line.
An echocardiogram (LOGIQe; GE Healthcare, Waukesha, WI) was performed under 1–3% isoflurane anesthesia in 100% O2 (1 L/min) at baseline and at 10 and 14 weeks after 6-OHDA. Measurements included heart rate, wall thickness, LV chamber diameter, fractional shortening (FS), velocity of blood through mitral and aortic valves, isovolumetric relaxation time, and aortic diameter.
Blood samples for plasma troponin I and catecholamines were obtained at baseline and at 1, 4, 10, and 14 weeks after toxin administration. Blood was collected in a K2 EDTA tube immediately mixed with 10% sodium metabisulfite (0.7%) and centrifuged. For plasma troponin I, samples were analyzed by an enhanced sensitivity immunometric immunoassay as per manufacture instructions (VITROS Immunodiagnostic Products). Plasma norepinephrine, dopamine and epinephrine and their deaminated metabolite dihydroxyphenylacetic acid (DOPAC) were assayed by high-performance liquid chromatography (HPLC) with electrochemical detection (ESA, Chelmsford, MA). Two animals (RH2318 and RH2316) had baseline catecholamines drawn under ketamine and medetomidine, an adrenergic agonist, and were removed from individual catecholamine analysis. A1.0 mL of plasma was analyzed using a coulometric electrochemical detector (Choulochem III; ESA, Chelmsford, MA). Every 1 L of mobile phase used to separate the catecholamines contained 13.8 g of sodium phosphate, 55 mg of 1-octane sulfonic acid, 55 mg of EDTA and 45 mL of acetonitrile at pH of 3.85 and filtered through a 0.22 µm GV filter under vacuum and pumped into the system at a rate of 0.6 mL/min producing a pressure approximately 69–71 bars. The electrodes were set at −250 and 380 mV. Peak heights were measured from the chromatograms and original concentrations of the plasma sample were determined by correcting for the incomplete recovery relative to the internal standards. Coefficients of variation between standards were at acceptable levels for norepinephrine (0.014), epinephrine (0.023), dopamine (0.015) and DOPAC (0.014). For statistical analysis, levels below detectable HPLC sensitivity, were considered as the threshold value (e.g.: epinephrine, 3.0 mg/mL; norepinephrine, 1.5 pg/mL; DOPAC and epinephrine assays were all within detectable ranges).
MHED was prepared by UW-Madison Medical Physics cyclotron facility using published methods [35]. Briefly, [11C]MeI was produced from in-target [11C]CH4 on a 16 MeV GE PETtrace using a Scansys automated chemistry module. Gas phase [11C]MeI labeled 15 mg metaraminol free base (ABX, Germany) in 300 µL 1∶2 DMSO∶DMF. The sealed vial was heated to 110°C for 5 minutes and diluted with 5 mL water for preparative reverse-phase HPLC purification (Econosil 5 µ, 8×300 mm, 3 mL/min 0.1 M Na2HPO4, with EtOH concentration increasing linearly from 0 to 10% over 600 s). The final product was millipore filtered for injection.
Monkeys underwent MHED PET under isoflurane anesthesia (1–3% in 100% O2, 1 L/min) at baseline and at 1, 4, 10, and 14 weeks after 6-OHDA. Animals were positioned supine in a Siemens HR+ PET scanner, using a custom-made foam positioning apparatus. After a 15 minute transmission scan, MHED was injected as an intravenous bolus (≤5.2 mCi) over 30 s. Dynamic PET images were obtained for 1 hour with conventionally increasing frame durations (6×30s, 3×60s, 2×120s, 10×300s).
Whole-blood tracer concentrations were obtained from a volume of interest in the upper part of the LV chamber. MHED uptake was quantified using the equilibrium distribution volume (DV) [36] of the LV tissue relative to whole blood. A DV value of 1 corresponds to no excess capacity in tissue relative to blood; DV – 1 thus provides a measure of the density of the nerve terminals that give the tissue its excess capacity. DV was evaluated in eight sectors in each of seven short-axis rings evenly spaced along the long axis of the heart. Data are presented as polar maps (apex of the heart at the center, base of the LV at the edge). Intersubject variation was observed at baseline in the apical and basal regions, probably reflecting normal patterns of innervation. Study of one animal (R01097) at the 10-week time point failed for technical reasons; uptake patterns at other time points were similar to those of the other four animals and were included in the visual assessments. Statistical assessments of PET data were performed with only the four remaining animals.
Individual subject global DV was calculated as an average over all eight sectors from polar rings 2 to 6 (total: 40 blocks) at baseline and at 1, 4, 10, and 14 weeks after lesion. These measures were averaged across animals to determine the group global DV at each timepoint. Individual MHED percent retention deficit was calculated for each of the 40 blocks at each time point as [(DVbaseline−1)−(DVtimepoint−1)]/(DVbaseline−1)×100 and averaged over blocks at each time point. These measures also were averaged across animals to obtain group MHED percent retention deficit at the four post-lesion time points.
All statistical analysis was performed using GraphPad Prism (version 5.0b, GraphPad Software). A P<0.05 was accepted as significant. Comparison over time of the different data sets (feces, weights, ECG intervals, echocardiogram data, troponin I and catecholamines levels) were done using repeated measures ANOVA and corrected with Bonferroni multiple comparison tests.
Changes over time of global DV mean and percent retention deficit were assessed by repeated-measures ANOVA and corrected with Tukey's multiple comparisons test. To assess regional variation, individual DV values in the four single sectors in the anterior, septal, inferior, and lateral directions were averaged over the five central rings (total: 5 blocks) to provide a single mean regional value at each time point. Group averaged regional DV values were analyzed with two-way repeated-measures ANOVA among all four regions over time and corrected with Bonferroni multiple comparisons test. Finally, Pearson correlations were performed between MHED uptake and other outcome measures (feces, ECG, blood assays and echocardiogram outputs) and between body weights and frequency of abnormal feces.
Sympathetic responses occurred immediately after each 6-OHDA injection, including increases in systolic and diastolic blood pressure (Figure 2A) and heart rate. Individual animal sensitivity and the tachyphylaxic effect of sympathetic responses to 6-OHDA dosing influenced the time interval between successive doses that ranged from 12 to 100 minutes with the longest period occurring after a cumulative dose of >1.5–3 mg/kg (Table 1; Figure 2B).
In three animals (R01098, R04094, RH2316), ECGs showed arrhythmic activity (e.g., ventricular premature contractions) when the cumulative dose reached >3 mg/kg, which delayed next dosing. Increasing the number of injections (while decreasing the amount of 6-OHDA per injection) and adjusting each dose according to the individual response minimized side effects and reduced time between consecutive doses.
Hematocrit levels were slightly elevated during toxin delivery; peak increases averaged 18.1%. Three animals (R01097, R01098, R04094) vomited during or directly after treatment. All animals produced pink-hued urine after 6-OHDA, probably because of oxidation of 6-OHDA; samples were negative for blood.
Group weekly weights showed body weight loss after 6-OHDA compared to baseline. The weight loss became statistically significant 4 weeks after lesion (12.26%) and remained stable over time (Table 2).
Soft feces or diarrhea were observed after 6-OHDA dosing but did not interfere with general and motor behavior as well as blood hematology and chemistry. The frequency of abnormal feces positively correlated with body weight loss (Pearson's correlation, r2 = 0.516, P<0.0001), in particular diarrhea (r2 = 0.3604, P = 0.0005) and soft feces (r2 = 0.4015, P = 0.0002).
ECGs after 6-OHDA all demonstrated normal sinus rhythm. There were no significant changes in PR, QRS and QTc intervals relative to baseline, despite QTc being considered an indicator of cardiac dysautonomia in Parkinson's disease (Table 3) [37], [38].
Echocardiograms revealed variable changes in FS, a measure of systolic performance following 6-OHDA (Table 4). Three animals had a decrease in FS throughout the study with the largest decrease being 43% from baseline. In two of these three animals, there was left ventricular dilation accompanying the systolic dysfunction. Two animals showed no apparent changes in chamber size or FS. There were no notable clinical consequences observed from FS reductions such as signs of heart failure. Evaluation of wall thickness, did not reveal significant changes associated to nonuniform wall innervation (Table 4).
Troponin I levels in four animals were not affected by 6-OHDA. One animal (R04094) had a fivefold increase at 1 week, suggestive of cardiac damage; levels returned to baseline over time.
Plasma catecholamine levels dramatically decreased 1 week after 6-OHDA and remained stable until necropsy (Table 5), with significant reduction in norepinephrine (F2,4 = 6.57, P = 0.012) and DOPAC (F2,4 = 17.20, P = 0.0005) levels. Decreases in dopamine and epinephrine did not reach statistical significance, probably because of individual variability at baseline.
Baseline DV values were uniform, regionally and across animals. DV values fell to ∼1 in all regions in all animals at 1 week after 6-OHDA, and slowly recovered over 3 months with considerable regional variation (Figure 3). Group-averaged global DV values at each time point after 6-OHDA were all significantly lower than at baseline (P<0.001), suggesting reduction of innervation 3 months after 6-OHDA. Similarly, group MHED percent retention deficit values varied significantly over time (F3,3 = 14.38, P<0.001), with reductions noted between 1 and 10 weeks (P<0.01), 1 and 14 weeks (P<0.001), and 4 and 14 weeks after toxin (P<0.05) but not between directly adjacent time points (Figure 4A). The continual increase in MHED uptake suggests slow but global recovery of innervation; however, significant changes were detected between 10 and 14 weeks after toxin challenge, suggesting stabilization of the lesion.
Statistical analysis demonstrated a main effect on each variable region (F3,48 = 5.93, P<0.05) and time (F4,48 = 231.4, P<0.001), in addition to the interaction effect (F12,48 = 2.986, P<0.01). Post hoc analysis between regions over time showed a significant difference between anterior and inferior regions at 10 (P<0.001) and 14 weeks (P<0.01), anterior and septal at 10 weeks (P<0.001), and inferior and lateral at 10 weeks (P<0.01), suggesting that 6-OHDA creates a heterogeneous lesion in the LV myocardium (Figure 4B).
Cardiac MHED uptake was significantly correlated with the natural logarithm of the blood concentrations of norepinephrine (Pearson's correlation; r2 = 0.44, P = 0.0097), dopamine (r2 = 0.596, P = 0.0012), epinephrine (r2 = 0.526, P = 0.0033), and DOPAC (r2 = 0.60, P = 0.0011) at similar time points (Figure 5). Additionally, frequency of abnormal feces significantly corresponded with MHED uptake (r2 = 0.4814, P = 0.0059). No significant correlations were found between MHED and troponin I or FS changes.
To our knowledge, this is the first study to characterize in vivo effects in the heart of systemic 6-OHDA to rhesus monkeys, and the first to track experimental cardiac denervation with MHED PET. The main findings include a nonuniform pattern of MHED uptake in the left myocardium, as well as decrease in circulating catecholamines, suggesting that this model mimics cardiac dysautonomia in PD.
The most common nonhuman primate model of PD involves the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [39]. Systemic dosing of MPTP produces a severe parkinsonian motor syndrome; however, effects in sympathetic denervation are temporary, as observed with 6-[18F]fluorodopamine PET imaging [40]. The other frequently used PD monkey model depends on direct intracerebral delivery of 6-OHDA, in order to bypass the blood–brain barrier and affect the nigrostriatal pathway without inducing peripheral effects [39]. Our method of intravenous delivery takes advantage of 6-OHDA catecholaminergic toxicity to develop a cardiac dysautonomia model.
The only previous report of systemic administration of 6-OHDA to monkeys was done in a single rhesus to compare the systemic effect of 6-OHDA to MPTP [40]. Yet, the authors did not describe the 6-OHDA method of administration and only provided a 6-[18F]fluorodopamine PET scan image and catecholamine levels 1 week after intoxication. Instead, we based our dosing regime on the reports of 6-OHDA administration to dogs, which described neurotoxin delivery over several hours to avoid clinical complications associated to the dopaminomimetic effects of the neurotoxin [31], [32]. Intravenous administration of 6-OHDA proved to require intensive monitoring and veterinary care to avoid hypertensive crisis and pulmonary edema. The adjustment of the dosing regimen to the individual animal response decreased the intensity of the symptoms and the time period needed for their normalization and ensured a safe recovery after the procedure.
Reduction in FS after 6-OHDA administration suggests an abnormality in cardiac contractility. These reductions were mild; even the lowest values were within published normal reference ranges [41], [42], and were not associated at the time with changes in wall thickness. It should be noted that normal ranges of echocardiogram parameters are not well established for macaque monkeys and the limited data are based on single recordings with different types of anesthesia. Because FS measurements are not well established for monkeys, we evaluated the percent change of FS over time for each individual animal. Three animals in this study had recurring declines in the change of FS following 6-OHDA, suggesting that the loss of noradrenergic cardiac innervation may produce reductions in left ventricular systolic function. The animal with the greatest decrease in FS (R04094) uniquely had increased troponin I levels 1 week after toxin suggesting injury to the myocardium. This was coupled with lack of recovery in the LV MHED uptake from 1 week to 4 weeks, perhaps demonstrating a more severe hypertensive response to 6-OHDA. The other animals did not exhibit elevated cardiac troponin I levels and had no or less of an initial decrease in FS accompanied by greater increases in total MHED uptake.
Noninvasive PET imaging of cardiac sympathetic innervation allowed us to monitor over time the cardiac tissue response to 6-OHDA challenge. We used equilibrium DV relative to whole blood to analyze uptake of MHED, because the uptake index conventionally used [20] declined rapidly as progressively later time windows were used for estimation. This behavior, and the success of an analysis method that assumes reversibility of the radioligand uptake, are at least partly due to progressive metabolism of MHED into compounds with no specific affinity to noradrenergic terminals. If blood metabolite analysis is not in the protocol, the DV estimates represent the most reproducible and least arbitrary way to use all the measured data to estimate a single number representing the uptake process. The use of DV – 1 to represent the innervation-specific part of the uptake is based on the observation that lesioning reduced the DV values to ∼1 in all regions in all animals.
Systemic administration of 6-OHDA induced loss of catecholaminergic innervation of the heart (greater loss in inferior LV myocardium), which persisted 3 months after toxin challenge. Although nerve terminals in all regions of the LV reacted similarly to 6-OHDA at 1 week, regional rates of recovery varied over time, with an average 51% remaining deficit after 3 months. The anterior region consistently recovered most rapidly and the inferior wall most slowly. Our findings resemble those in PD: MHED PET established loss in sympathetic myocardial innervation [19] and 6-[18F]fluorodopamine imaging suggested preservation in septal [18] and anterior [6] walls of the LV. The nonuniform pattern of MHED uptake may be caused by regional differences in blood perfusion that affected the distribution or metabolism of the neurotoxin. Another possibility is that the subpopulation of ganglionic cells innervating the area has a greater sensitivity to 6-OHDA. It could be argued that the regional recovery was due to re-growth of cardiac muscle tissue, instead of reinnervation. Yet, results from the echocardiogram showed no significant differences in posterior or anterior wall thickness in either diastole or systole. Bai and colleagues [43] have reported recovery of cardiac sympathetic nerves following subcutaneous delivery of 6-OHDA insult in rats. In addition, sympathetic reinnervation has been described after heart transplantation, suggesting that the system has a certain plasticity that could be exploited for regenerative or neuroprotective treatments [44], [45], [46]. Increasing animal numbers would help further define regional differences in LV MHED uptake. Further investigation of the mechanisms of regional loss is needed, as it may facilitate the identification of possible therapeutic targets.
The timeline of our experimental design was based on our previous experience with administration of neurotoxins in the central nervous system of nonhuman primates. For example, an observation period of 3 months after MPTP challenge allows for the dopaminergic nigral cell neurodegeneration to be completed and defines a stable syndrome [33], [47], [48]. Lack of significant changes in MHED uptake between 10 and 14 weeks suggests that as predicted, by 3 months the catecholaminergic lesion was stabilized and recovery mechanisms were completed. The characterization of the timeline for recovery and stabilization of the lesion in this model will be helpful when designing a study to test disease-modifying strategies for the heart. Follow-up experiments with endpoints exceeding 14 weeks after toxin administration would further confirm that 6-OHDA produces a stable lesion in the LV.
Dopamine, epinephrine, norepinephrine, and DOPAC circulating levels were significantly decreased after 6-OHDA, which indicates that the neurotoxin affected peripheral catecholaminergic sources such as adrenal medulla, facilitating onset of cardiac dysautonomia. The positive correlation found between catecholamine levels and cardiac MHED uptake further suggests a similar toxic effect of 6-OHDA in different peripheral catecholaminergic cells. The drop in circulating catecholamines did not seem to affect animal health, probably because of adaptive sympathetic presynaptic supersensitivity [49]. Studies in dogs did not find changes in catecholamine levels, suggesting a species difference in the sensitivity to neurotoxin effects [32].
Cardiac dysautonomia in PD patients is clinically characterized by orthostatic hypotension, an increase in corrected QT intervals (QTc), and reductions in heart rate variability [6], [7], [8], [9], [10], [52], [53]. The presence of these symptoms was not confirmed in this study, because they are detected in an awake state and the cardiac and blood pressure evaluations were performed under anesthesia. Nonuniform cardiac innervation, affects cardiac repolarization and has been associated with arrhythmias [6], [15]. Future preclinical experiments using telemetric measurements of heart rate, blood pressure, and locomotive activity [50], [51] will allow their identification in awake animals. Injections of vasoactive pharmaceuticals such as phenylephrine or sodium nitroprusside may also help characterize the cardiovascular response.
The monkeys presented abnormal feces and weight loss that became significant 4 weeks after 6-OHDA. The frequency of loose stools and diarrhea correlated with the amount of weight loss. Supplementation of feedings with chow soaked in protein-enriched drink (standard practice in our facility for animals loosing weight) enticed feeding, increased fluid intake and helped prevent further weight loss. The abnormal feces could have been the result from stress (each animal underwent procedures at least monthly), but their presence were also described in 6-OHDA-treated dogs that did not undergo those evaluations, suggesting an effect of 6-OHDA in the gastrointestinal tract [31]. The enteric nervous system consists of dopaminergic neurons (myenteric and submucosal plexus), which are potentially susceptible to the toxic effects of systemic 6-OHDA [54]. In that regard, rats treated with 6-OHDA have decreased TH mRNA levels in the duodenum [55]. Reduced sympathetic innervation of the intestinal tract can lead to bowel dysmotility and, therefore, abnormal feces.
Collectively, the changes in cardiac innervation, catecholamine levels and feces suggest that systemic dosing of 6-OHDA affects multiple vulnerable catecholaminergic peripheral cells, and this property can be applied to model dysautonomias in nonhuman primates. Similar to CNS neurotoxic models, a 6-OHDA-induced dysautonomia model presents limitations like acute onset and the risk for spontaneous recovery [39]. Intravenous administration of 6-OHDA does not induce a PD motor syndrome, therefore a comprehensive PD model would require supplementation with systemic MPTP or direct intracerebral dosing of 6-OHDA to induce dopaminergic nigral cell loss. Postmortem analysis and quantification of catecholaminergic cell and terminal loss in susceptible tissues, as well as evaluation of pathologies typical of PD–related neurodegeneration, such as inflammatory cell response and intracytoplasmic accumulation of alpha synuclein [56], are warranted to further characterize the effect of 6-OHDA in peripheral catecholaminergic cells.
To conclude, systemic administration of 6-OHDA to rhesus monkeys mimics features of cardiac dysautonomia in PD that can be tracked and mapped in vivo using PET imaging. We hope that these results will facilitate model development to study this symptom and to identify new therapeutic alternatives.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This project was supported in part by the Clinical and Translational Science Award (CTSA) program, previously through the National Center for Research Resources (NCRR) grant 1UL1RR025011, and now by the National Center for Advancing Translational Sciences (NCATS), grant 9U54TR000021. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health (NIH). The authors also acknowledge the support of WNPRC NIH base grant P51RR000167, and the faculty startup funds from the University of Wisconsin Graduate School, Medical School, and Medical Physics Department. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We thank Victoria Carter and Michael Dobbert for excellent technical assistance.
1. | Jost WH. Year: 2003Autonomic dysfunctions in idiopathic Parkinson's disease.J Neurol250Suppl 1I283012761632 |
2. | Martignoni E,Pacchetti C,Godi L,Micieli G,Nappi G. Year: 1995Autonomic disorders in Parkinson's disease.J Neural TransmSuppl 451119 |
3. | Rajput AH,Rozdilsky B. Year: 1976Dysautonomia in Parkinsonism: a clinicopathological study.J Neurol Neurosurg Psychiatry3910921100188990 |
4. | Martinez-Martin P,Schapira AH,Stocchi F,Sethi K,Odin P,et al. Year: 2007Prevalence of nonmotor symptoms in Parkinson's disease in an international setting; study using nonmotor symptoms questionnaire in 545 patients.Mov Disord221623162917546669 |
5. | Chaudhuri KR,Martinez-Martin P,Schapira AH,Stocchi F,Sethi K,et al. Year: 2006International multicenter pilot study of the first comprehensive self-completed nonmotor symptoms questionnaire for Parkinson's disease: the NMSQuest study.Mov Disord2191692316547944 |
6. | Goldstein DS. Year: 2003Dysautonomia in Parkinson's disease: neurocardiological abnormalities.Lancet Neurol266967614572735 |
7. | Post KK,Singer C,Papapetropoulos S. Year: 2008Cardiac denervation and dysautonomia in Parkinson's disease: a review of screening techniques.Parkinsonism Relat Disord1452453118486523 |
8. | Pursiainen V,Haapaniemi TH,Korpelainen JT,Huikuri HV,Sotaniemi KA,et al. Year: 2002Circadian heart rate variability in Parkinson's disease.J Neurol2491535154012420094 |
9. | Mathias CJ. Year: 1998Cardiovascular autonomic dysfunction in parkinsonian patients.Clin Neurosci515316610785842 |
10. | Oka H,Mochio S,Onouchi K,Morita M,Yoshioka M,et al. Year: 2006Cardiovascular dysautonomia in de novo Parkinson's disease.J Neurol Sci241596516325862 |
11. | Goldstein DS,Holmes C,Li ST,Bruce S,Metman LV,et al. Year: 2000Cardiac sympathetic denervation in Parkinson disease.Ann Intern Med13333834710979878 |
12. | Takatsu H,Nishida H,Matsuo H,Watanabe S,Nagashima K,et al. Year: 2000Cardiac sympathetic denervation from the early stage of Parkinson's disease: clinical and experimental studies with radiolabeled MIBG.J Nucl Med41717710647607 |
13. | Braune S,Reinhardt M,Bathmann J,Krause T,Lehmann M,et al. Year: 1998Impaired cardiac uptake of meta-[123I]iodobenzylguanidine in Parkinson's disease with autonomic failure.Acta Neurol Scand973073149613560 |
14. | Taki J,Nakajima K,Hwang EH,Matsunari I,Komai K,et al. Year: 2000Peripheral sympathetic dysfunction in patients with Parkinson's disease without autonomic failure is heart selective and disease specific. email@example.com J Nucl Med2756657310853813 |
15. | Podrid PJ,Fuchs T,Candinas R. Year: 1990Role of the sympathetic nervous system in the genesis of ventricular arrhythmia.Circulation82I1031131973640 |
16. | Tipre DN,Goldstein DS. Year: 2005Cardiac and extracardiac sympathetic denervation in Parkinson's disease with orthostatic hypotension and in pure autonomic failure.J Nucl Med461775178116269589 |
17. | Goldstein DS,Sharabi Y,Karp BI,Bentho O,Saleem A,et al. Year: 2007Cardiac sympathetic denervation preceding motor signs in Parkinson disease.Clin Auton Res1711812117334896 |
18. | Li ST,Dendi R,Holmes C,Goldstein DS. Year: 2002Progressive loss of cardiac sympathetic innervation in Parkinson's disease.Ann Neurol5222022312210793 |
19. | Berding G,Schrader CH,Peschel T,van den Hoff J,Kolbe H,et al. Year: 2003[N-methyl 11C]meta-Hydroxyephedrine positron emission tomography in Parkinson's disease and multiple system atrophy.Eur J Nucl Med Mol Imaging3012713112483420 |
20. | Raffel DM,Koeppe RA,Little R,Wang CN,Liu S,et al. Year: 2006PET measurement of cardiac and nigrostriatal denervation in Parkinsonian syndromes.J Nucl Med471769177717079809 |
21. | Orimo S,Ozawa E,Nakade S,Sugimoto T,Mizusawa H. Year: 1999(123)I-metaiodobenzylguanidine myocardial scintigraphy in Parkinson's disease.J Neurol Neurosurg Psychiatry6718919410406987 |
22. | King AE,Mintz J,Royall DR. Year: 2010Meta-analysis of 123I-MIBG cardiac scintigraphy for the diagnosis of Lewy body-related disorders.Mov Disord261218122421480373 |
23. | Suzuki M,Urashima M,Oka H,Hashimoto M,Taira K. Year: 2007Cardiac sympathetic denervation in bradykinesia-dominant Parkinson's disease.Neuroreport181867187018090328 |
24. | Goldstein DS,Eldadah B,Sharabi Y,Axelrod FB. Year: 2008Cardiac sympathetic hypo-innervation in familial dysautonomia.Clin Auton Res1811511918498023 |
25. | Orimo S,Amino T,Itoh Y,Takahashi A,Kojo T,et al. Year: 2005Cardiac sympathetic denervation precedes neuronal loss in the sympathetic ganglia in Lewy body disease.Acta Neuropathol10958358815933869 |
26. | Orimo S,Oka T,Miura H,Tsuchiya K,Mori F,et al. Year: 2002Sympathetic cardiac denervation in Parkinson's disease and pure autonomic failure but not in multiple system atrophy.J Neurol Neurosurg Psychiatry7377677712438492 |
27. | Amino T,Orimo S,Itoh Y,Takahashi A,Uchihara T,et al. Year: 2005Profound cardiac sympathetic denervation occurs in Parkinson disease.Brain Pathol15293415779234 |
28. | Emborg ME. Year: 2004Evaluation of animal models of Parkinson's disease for neuroprotective strategies.J Neurosci Methods13912114315488225 |
29. | Porter CC,Totaro JA,Burcin A. Year: 1965The relationship between radioactivity and norepinephrine concentrations in the brains and hearts of mice following administration of labeled methyldopa or 6-hydroxydopamine.J Pharmacol Exp Ther15017225853698 |
30. | Porter CC,Totaro JA,Stone CA. Year: 1963Effect of 6-hydroxydopamine and some other compounds on the concentration of norepinephrine in the hearts of mice.J Pharmacol Exp Ther14030831613972348 |
31. | Gauthier P,Nadeau R,De Champlain J. Year: 1972Acute and chronic cardiovascular effects of 6-hydroxydopamine in dogs.Circ Res312072174340389 |
32. | Goldstein DS,Grossman E,Tamrat M,Chang PC,Eisenhofer G,et al. Year: 1991Positron emission imaging of cardiac sympathetic innervation and function using 18F-6-fluorodopamine: effects of chemical sympathectomy by 6-hydroxydopamine.J Hypertens94174231649861 |
33. | Emborg ME,Ma SY,Mufson EJ,Levey AI,Taylor MD,et al. Year: 1998Age-related declines in nigral neuronal function correlate with motor impairments in rhesus monkeys.J Comp Neurol4012532659822152 |
34. | Emborg ME,Moirano J,Schafernak KT,Moirano M,Evans M,et al. Year: 2006Basal ganglia lesions after MPTP administration in rhesus monkeys.Neurobiol Dis2328128916766201 |
35. | Rosenspire KC,Haka MS,Van Dort ME,Jewett DM,Gildersleeve DL,et al. Year: 1990Synthesis and preliminary evaluation of carbon-11-meta-hydroxyephedrine: a false transmitter agent for heart neuronal imaging.J Nucl Med31132813342384800 |
36. | Logan J,Fowler JS,Volkow ND,Wolf AP,Dewey SL,et al. Year: 1990Graphical analysis of reversible radioligand binding from time-activity measurements applied to [N-11C-methyl]-(-)-cocaine PET studies in human subjects.J Cereb Blood Flow Metab107407472384545 |
37. | Ishizaki F,Harada T,Yoshinaga H,Nakayama T,Yamamura Y,et al. Year: 1996[Prolonged QTc intervals in Parkinson's disease–relation to sudden death and autonomic dysfunction].No To Shinkei484434488672303 |
38. | Deguchi K,Sasaki I,Tsukaguchi M,Kamoda M,Touge T,et al. Year: 2002Abnormalities of rate-corrected QT intervals in Parkinson's disease-a comparison with multiple system atrophy and progressive supranuclear palsy.J Neurol Sci199313712084439 |
39. | Emborg ME. Year: 2007Nonhuman primate models of Parkinson's disease.ILAR J4833935517712221 |
40. | Goldstein DS,Li ST,Holmes C,Bankiewicz K. Year: 2003Sympathetic innervation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine primate model of Parkinson's disease.J Pharmacol Exp Ther30685586012805479 |
41. | Sleeper MM,Gaughan JM,Gleason CR,Burkett DE. Year: 2008Echocardiographic reference ranges for sedated healthy cynomolgus monkeys (Macaca fascicularis).J Am Assoc Lab Anim Sci47222518210994 |
42. | Gross DR. Year: 2009Normal Cardiac Function Parameters.Gross DRAnimal Models in Cardiovascular Research. 3 edNew YorkSpringer5564 |
43. | Bai J,Ren C,Hao W,Wang R,Cao JM. Year: 2008Chemical sympathetic denervation, suppression of myocardial transient outward potassium current, and ventricular fibrillation in the rat.Can J Physiol Pharmacol8670070918841175 |
44. | Kaye DM,Esler M,Kingwell B,McPherson G,Esmore D,et al. Year: 1993Functional and neurochemical evidence for partial cardiac sympathetic reinnervation after cardiac transplantation in humans.Circulation88111011188353872 |
45. | Bengel FM,Ueberfuhr P,Ziegler SI,Nekolla S,Reichart B,et al. Year: 1999Serial assessment of sympathetic reinnervation after orthotopic heart transplantation. A longitudinal study using PET and C-11 hydroxyephedrine.Circulation991866187110199884 |
46. | Buendia-Fuentes F,Almenar L,Ruiz C,Vercher JL,Sanchez-Lazaro I,et al. Year: 2011Sympathetic reinnervation 1 year after heart transplantation, assessed using iodine-123 metaiodobenzylguanidine imaging.Transplant Proc432247224821839246 |
47. | Emborg ME,Moirano J,Raschke J,Bondarenko V,Zufferey R,et al. Year: 2009Response of aged parkinsonian monkeys to in vivo gene transfer of GDNF.Neurobiol Dis |
48. | Swanson CR,Joers V,Bondarenko V,Brunner K,Simmons HA,et al. Year: 2011The PPAR-gamma agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys.J Neuroinflammation89121819568 |
49. | Nadeau RA,de Champlain J,Tremblay GM. Year: 1971Supersensitivity of the isolated rat heart after chemical sympathectomy with 6-hydroxydopamine.Can J Physiol Pharmacol4936445572416 |
50. | Slack K,Billing R,Matthews S,Allbutt HN,Einstein R,et al. Year: 2010Subtle cardiovascular dysfunction in the unilateral 6-hydroxydopamine-lesioned rat.Parkinsons Dis201042781020976085 |
51. | Sakata M,Sei H,Toida K,Fujihara H,Urushihara R,et al. Year: 2002Mesolimbic dopaminergic system is involved in diurnal blood pressure regulation.Brain Res92819420111844487 |
52. | Goldstein DS. Year: 2006Orthostatic hypotension as an early finding in Parkinson's disease.Clin Auton Res16465416477495 |
53. | Valappil RA,Black JE,Broderick MJ,Carrillo O,Frenette E,et al. Year: 2010Exploring the electrocardiogram as a potential tool to screen for premotor Parkinson's disease.Mov Disord252296230320976736 |
54. | Cersosimo MG,Benarroch EE. Year: 2008Neural control of the gastrointestinal tract: implications for Parkinson disease.Mov Disord231065107518442139 |
55. | Kawamura M,Schwartz JP,Nomura T,Kopin IJ,Goldstein DS,et al. Year: 1999Differential effects of chemical sympathectomy on expression and activity of tyrosine hydroxylase and levels of catecholamines and DOPA in peripheral tissues of rats.Neurochem Res2425329973233 |
56. | Braak H,Braak E. Year: 2000Pathoanatomy of Parkinson's disease.J Neurol247Suppl 2II31010991663 |
Table 1 6-OHDA dosing scheme (mg/kg) for each individual animal.
Injection number | R01097 | R01098 | R04094 | RH2316 | RH2318 |
1 | 0.50 | 0.50 | 0.50 | 0.49 | 0.50 |
2 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
3 | 1.50 | 0.62 | 1.50 | 2.30 | 1.50 |
4 | 2.00 | 0.88 | 2.00 | 0.85 | 2.00 |
5 | 4.00 | 2.00 | 4.00 | 5.00 | 4.00 |
6 | 15.00 | 4.00 | 15.00 | 10.00 | 10.00 |
7 | 26.00 | 10.00 | 26.00 | 11.84 | 10.00 |
8 | 10.00 | 18.52 | 21.00 | ||
9 | 21.00 | ||||
Total | 50.00 | 50.00 | 50.00 | 50.00 | 50.00 |
The rhesus monkeys received 7–9 injections of 6-OHDA solution in one session, accumulating to a total final dose of 50 mg/kg. Dosing and timeline of injections varied based on the individual reaction to 6-OHDA. The normalization (return to baseline measures) of vital signs, ECG, heart rate, and blood pressure was used to define when the next dose could be given.
Table 2 Weekly animal weights (kg) and average group % weight loss before and after 6-OHDA dosing.
Animal ID | Baseline | 1 | 2 | 3 | 4 | 8 | 10 | 11 | 12 | 13 | 14 |
Average (kg) | 8.39 | 7.85 | 7.60 | 7.67 | 7.37** | 7.39** | 7.36** | 7.50* | 7.30** | 7.44* | 7.35** |
Average group % loss | 6.72 | 9.25 | 8.40 | 12.26 | 12.32 | 12.78 | 10.84 | 13.49 | 11.92 | 12.95 |
A significant loss of weight was found starting at four weeks post 6-OHDA challenge and persisting throughout the study.
Table 3 Electrocardiogram (ECG) measurements at baseline, 1, 4, 10 and 14 weeks after 6-OHDA for each animal.
Animal ID | Time | HR (bpm) | PR (ms) | QRS (ms) | QT (ms) | QRS axis | ΔQTc |
RH2316 | Baseline | 140 | 80 | 45 | 230 | 84 | |
1 wk | 112 | 90 | 77 | 296 | 65 | 53.10 | |
4 wk | 112 | 90 | 40 | 240 | 110 | −23.40 | |
10 wk | 130 | 105 | 50 | 250 | 110 | 16.70 | |
14 wk | 135 | 90 | 48 | 242 | 110 | 11.70 | |
RH2318 | Baseline | 130 | 78 | 50 | 260 | 45 | |
1 wk | 125 | 80 | 48 | 265 | 30 | −0.21 | |
4 wk | 142 | 95 | 40 | 240 | 45 | −13.51 | |
10 wk | 148 | 82 | 38 | 280 | 45 | 57.09 | |
14 wk | 135 | 100 | 44 | 285 | 45 | 44.79 | |
R01097 | Baseline | 132 | 90 | 50 | 240 | 50 | |
1 wk | 129 | 90 | 45 | 280 | 45 | 54.60 | |
4 wk | 165 | 85 | 43 | 240 | 45 | 42.00 | |
10 wk | 140 | 90 | 47 | 235 | 45 | 33.40 | |
14 wk | 137 | 100 | 46 | 245 | 45 | 6.70 | |
R01098 | Baseline | 120 | 92 | 45 | 295 | 45 | |
1 wk | 135 | 95 | 54 | 270 | 80 | −12.20 | |
4 wk | 145 | 90 | 55 | 290 | 45 | 33.60 | |
10 wk | 138 | 98 | 54 | 269 | 45 | 20.20 | |
14 wk | 134 | 89 | 51 | 300 | 45 | 21.70 | |
R04094 | Baseline | 140 | 98 | 48 | 250 | 65 | |
1 wk | 120 | 104 | 50 | 315 | 65 | 63.60 | |
4 wk | 140 | 100 | 51 | 260 | 85 | 15.30 | |
10 wk | 132 | 90 | 54 | 280 | 90 | −9.20 | |
14 wk | 145 | 90 | 50 | 250 | 85 | 31.10 |
QTc was calculated using the Bazett's formula as QT interval/sqrt (RR interval), where the RR interval was calculated 60/HR. ΔQTc was calculated as QTc of each time point minus baseline. No significant differences were found in PR, QRS and QTc intervals obtained in anesthetized animals. HR, heart rate. QTc, corrected QT interval.
Table 4 Echocardiogram measurements at baseline, 10 and 14 weeks after 6-OHDA for each animal.
Animal ID | Time | AWd (cm) | AWs (cm) | PWd (cm) | PWs (cm) | LVDd (cm) | LVDs (cm) | FS | FS (% change from baseline) |
RH2316 | Baseline | 0.48 | 0.86 | 0.44 | 1.01 | 2.10 | 0.94 | 55% | |
10 wk | 0.44 | 0.85 | 0.44 | 0.95 | 2.20 | 1.08 | 51% | −8% | |
14 wk | 0.45 | 0.96 | 0.45 | 0.96 | 2.22 | 0.96 | 57% | +3% | |
RH2318 | Baseline | 0.47 | 0.88 | 0.50 | 1.08 | 2.35 | 1.28 | 46% | |
10 wk | 0.48 | 0.76 | 0.52 | 0.82 | 2.37 | 1.75 | 26% | −43% | |
14 wk | 0.48 | 0.86 | 0.49 | 0.95 | 2.52 | 1.60 | 37% | −20% | |
R01097 | Baseline | 0.47 | 0.81 | 0.48 | 0.75 | 2.01 | 0.94 | 53% | |
10 wk | 0.49 | 0.82 | 0.47 | 0.74 | 2.17 | 1.10 | 49% | −7% | |
14 wk | 0.51 | 0.89 | 0.48 | 0.81 | 2.19 | 1.35 | 38% | −28% | |
R01098 | Baseline | 0.46 | 0.93 | 0.48 | 0.78 | 2.06 | 1.16 | 44% | |
10 wk | 0.45 | 0.77 | 0.45 | 0.72 | 1.97 | 1.16 | 41% | −6% | |
14 wk | 0.44 | 0.82 | 0.40 | 0.72 | 1.98 | 1.11 | 44% | +1% | |
R04094 | Baseline | 0.46 | 0.77 | 0.49 | 0.82 | 2.16 | 1.19 | 45% | |
10 wk | 0.45 | 0.86 | 0.42 | 0.86 | 2.15 | 1.12 | 48% | +7% | |
14 wk | 0.48 | 0.87 | 0.42 | 0.71 | 2.02 | 1.32 | 35% | −23% |
FS is calculated as [(LVDd – LVDs)/LVDd]×100. Three animals experienced large decreases in FS compared to baseline, but there was no significant change in left ventricle diameter or in anterior and posterior wall thickness. This suggests that the increased luminal dimensions of the left ventricle were not due to the loss of cardiac muscle. AWd, anterior wall thickness in diastole. AWs, anterior wall thickness in systole. PWd, posterior wall thickness in diastole. PWs, posterior wall thickness in systole. LVDd, left ventricle diameter in diastole. LVDs, left ventricle diameter in systole. FS, fractional shortening.
Table 5 Circulating plasma catecholamine levels (pg/mL) at baseline (0) and at 1, 4, 10, and 14 weeks after 6-OHDA in three animals.
Animal ID | Time | Norepinephrine pg/mL (ln)* | Epinephrine pg/mL (ln) | Dopamine pg/mL (ln) | DOPAC pg/mL (ln)** |
1 | 3.61 (1.28) | 3.83 (1.34) | 1.74 (0.56) | 26.48 (3.28) | |
4 | 4.73 (1.55) | 4.08 (1.41) | 1.26 (0.23) | 13.52 (2.60) | |
10 | 3.85 (1.35) | nd | 1.31 (0.27) | 14.46 (2.67) | |
14 | nd | nd | 2.77 (1.02) | 21.29 (3.06) | |
1 | 4.18 (1.43) | 3.20 (1.16) | 2.02 (0.70) | 14.99 (2.71) | |
4 | 4.50 (1.50) | 6.02 (1.80) | 1.36 (0.31) | 12.50 (2.53) | |
10 | nd | 4.11 (1.41) | 3.55 (1.27) | 12.13 (2.50) | |
14 | 4.81 (1.57) | 5.24 (1.66) | 2.82 (1.04) | 17.15 (2.84) | |
1 | 4.25 (1.45) | 1.87 (0.63) | 3.57 (1.27) | 16.82 (2.82) | |
4 | 3.91 (1.36) | 3.35 (1.21) | 1.03 (0.03) | 16.21 (2.79) | |
10 | 2.26 (0.81) | 3.43 (0.81) | 2.49 (0.91) | 19.04 (2.95) | |
14 | 4.39 (1.48) | nd | 7.12 (1.96) | 21.47 (3.07) |
The values in parentheses are the natural logarithms of the concentration values and used to graph correlations in Figure 5. For statistical analysis, non-detectable levels (nd) levels were considered as the lowest detection level with 1.5 pg/mL for norepinephrine and 3.0 pg/mL for epinephrine. Post hoc analysis with Bonferroni multiple comparisons detected significance at all timepoints compared to baseline for norepinephrine and DOPAC (*P<0.05, ** P<0.01).
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You absolutely love Curve, the English band from the early 90s that sounded so perfect, even if you cannot figure out why there's a sheep in the studio. You would not have had the band Garbage later without Curve and Tony Halliday.
Wednesday, June 10, 2009
2 comments:
- Lynda said...
Is this some sort of hypnotism??
- June 10, 2009 at 6:05 PM
- Captain Awesome said...
If you don't listen to Ike Reilly you should. This has nothing to do with this post it's just a music recommendation for you.
- June 10, 2009 at 10:35 PM
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LOL! My dog does! *snicker* However, I think that bulldog is faking... he fell asleep! ;)
Oh my goodness! This is so very, very adorable!! I absolutely love it (:
I don't have a dog that reads, but I have a cat that writes. I'm kind of pressuring her to be the next J. K. Rowling. But, you know, with cats.
Melissa - lol! My dog doesn't read, but he watches TV. Lazy ;)Rubita - I need to see a picture of that cuteness :D
Haha omg so cute. I wish my dog would do this but he wouldn't dare sit still for me to take a picture.
aww way too adorable. My puppy loves to watch Dogs on the Tv
Awww so cute. My favourite is the bull terrier :)
Haha, love it. Boris needs to start a book club.
Awww, this post is so full of cuteness!
Aww! Too cute!
Thank you Belle for posting; cute pictures in deed :) We featured some of them on our Facebook page, because these fit perfectly into what we were looking for :) Have a great day,
Hello! We LOVE the 7th image down and would like to feature it in an article on our site. Please get in touch for more details - email@example.com. Thanks!


text:
"The fecal immunochemical test (FIT) is highly effective for annual colorectal cancer screening programs for average-risk patients, according to results of a study published online January 26 in the Annals of Internal Medicine.
Read the Fecal Immunochemical Test Screening Effective at 4 Years article »
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What is cyclophosphamide (Cytoxan)?
Cyclophosphamide is a cancer medication that interferes with the growth and spread of cancer cells in the body.
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- Cytoxan Drug Interactions Center: cyclophosphamide oral
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Cytoxan - User Reviews
Report Problems to the Food and Drug Administration
You are encouraged to report negative side effects of prescription drugs to the FDA. Visit the FDA MedWatch website or call 1-800-FDA-1088.
Get the latest treatment options.


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This past week I accompanied my granddaughter, Maddie, a senior at Spencer County High School, to Signature HealthCare in Taylorsville. A school project encouraged her to make this visit.
While there, Maddie shared her sweet smile, love, concern and best wishes by giving a carnation and a Happy St. Patrick's Day note to each resident. She was welcomed with much love from each one she visited.
If you currently subscribe or have subscribed in the past to the Spencer Magnet, then simply find your account number on your mailing label and enter it below.
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Proof-reading: on-going project
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text: For most of the area in and around Haliburton County, the stated frost free date is between May 2 and May 31. This wide a discrepancy can be disastrous as aggressive growers may plant and transplant starting before the May 1st date gambling that frost will not occur when the young shoots appear above the soil. There are many ways to advance the date: some of which are to grow the plants in greenhouses, use cold frame protection of beds and also use raised bed gardening techniques. In recent years the frost free date has been advancing towards the first of May from the traditional May 24th date used in the past. Watching weather reports closely and being prepared to cover the garden in the event of frost will help to mitigate loss through frost if planting early. There are many vegetables that prefer the colder weather and may be safely begun as soon as the soil is workable. These include lettuce, peas, potatoes, and onions among others. David Hinks, Ottawa No, Victoria Day is not sacred. In fact, long before Victoria Day, over half of my vegetable garden is planted. By then I am even harvesting and eating some of the early crops, such as lettuce, radish and spinach ... Shane Rajapakse Etobicoke Crop rotation is not just for the farmers it can be put to use in small plots of land as well... |


text: Made using hand woven cotton fabric, this bed cover is perfect for your bed room. The self-weave featured on this bed cover add beauty to your luxury room. Highlight the look of this bedcover by adding contrast colour cushion from our range. This can also be a good option for gifting


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Karisma Kapoor
From Wikipedia, the free encyclopedia
Karisma Kapoor | |
Photograph of Karisma Kapoor
Born | Karishma Kapoor June 25, 1974 Mumbai, Maharashtra, India |
Occupation | Actress |
Years active | 1991-2003 |
Spouse(s) | Sanjay Kapur (2003-present) |
Karisma Kapoor (also spelled as Karishma Kapoor), is an Indian actress. She was born on 25 June 1974 in Mumbai, Maharashtra, India. Karishma Kapoor acts in Hindi language movies. She has won a Filmfare Best Actress Award. She also won a Filmfare Best Supporting Actress Award and a National Film Award for Best Supporting Actress.
Early life[change | change source]
She is the sister of actress Kareena Kapoor. She is the daughter of Bollywood Actors Randhir Kapoor and Babita. She is the first-cousin of actor Ranbir Kapoor. Kapoor studied at the Cathedral and John Connon School in Mumbai until Grade Six.
Personal life[change | change source]
She married Sanjay in 2003 and they have two children.


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AlsaModularSynth is a realtime modular synthesizer and effect
processor. It features: * MIDI controlled modular software
synthesis. * Realtime effect processing with capture from e.g.
"Line In" or "Mic In". * Full control of all synthesis and
effect parameters via MIDI. * Integrated LADSPA Browser with
search capability. * JACK Support.
License | Verified by | Verified on | Notes |
GPLv2 | Debian | 20 March 2013 |
Leaders and contributors
Resources and communication
Audience | Resource type | URI |
Download | http://sourceforge.net/project/showfiles.php?group_id=69130 |
Software prerequisites
Permission is granted to copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1.3 or any later version published by the Free Software Foundation; with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts. A copy of the license is included in the page "GNU Free Documentation License".
The copyright and license notices on this page only apply to the text on this page. Any software or copyright-licenses or other similar notices described in this text has its own copyright notice and license, which can usually be found in the distribution or license text itself.


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We have over 4,000 timeshare resorts for owners to post their week for rent and/or sale. Vacationers can rent or buy a week directly from the owner. Owners - to find your vacation resort click the RCI timeshare or II timeshare resort below. Do a quick search here. Vacationers - for discount vacation rentals. Rent a vacation condo at a discount directly from the Owner. Start your search here. |
USA resorts are below: |


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Standing 4'3", and possessed of a nails-on-the-blackboard speaking voice, actress Zelda Rubinstein specialized in eccentric characterizations. She was appropriately cast as one of the Little People in her first film, Under the Rainbow (1981). Her best-remembered screen role was Tangina, the disastrously self-assured exorcist in the first two Poltergeist films. Zelda's declaration that "This house is clean" has to be the most famous speaking-too-soon miscalculations in movie history. She was later seen in oddball minor roles as domineering mamas, witches, snoops and gamblers. TV fans know Zelda Rubinstein best as Ginny Weedon on the weekly cult favorite Picket Fences (1992-95). In January of 2010, after a long run-in with health issues, Rubinstein passed away at a respiratory hospital in Los Angeles.
— Hal Erickson, Rovi
— Hal Erickson, Rovi
Provided by Rovi


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OK, the rumors are correct again for both the iPad mini and the new revamped iPad. The iPad mini, in my opinion, is a smaller size iPad 2 (of course with slightly better front facing and rear cameras and new lightning port) and the new revamped iPad or 4th Gen iPad is just an iPad 3s.
Pre-order for both will start from 26th October 2012 and they will be available from 2nd November for WiFi only and 2 weeks later for cellular version (with LTE – not sure if it is the same configuration as iPhone 5).
Singapore will be getting the iPad mini on the 2nd November 2012 (WiFi only).
The iPad mini has a 7.9-inch screen and based on Apple, it will have the same resolution as the iPad 2 so that developers will not need to do anything on their apps for the iPad mini. Sound logical.
The weight and dimension of iPad mini: -
- Height: 7.87 inches (200 mm)
- Width: 5.3 inches (134.7 mm)
- Depth: 0.28 inch (7.2 mm)
- Weight (Wi-Fi): 0.68 pound (308 g)
- Weight (Wi-Fi + Cellular): 0.69 pound (312 g)
Click Here to Check Out the iPad mini
I have put together some easy comparison on the iPad family (I focus only on the changes): iPad mini, iPad 2, new iPad
Click Here for the full comparison table
Overall, I think the iPad mini, with the pricing, the specs and the ecosystem, is a killer for Nexus 7, Kindle HD and basically any 7-inchish tablets around.
However, on the revamped new iPad, I feel that Apple is rushing it out to put a difference (superiority) on the iPad mini with a new processor, better front facing camera and of course the new lightning port.
I wonder what would Steve Jobs has done if he is still around.
Of course, that was 2 years ago and things change. So, he might have made the same decision.
Here is the Press Release


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Brookfield, WI, USA < change city >
Bands (39)
Florists (39)
Cakes (36)
Caterers (34)
DJs (34)
Wedding Day Beauty (19)
Wedding Fashion (13)
Limos (11)
Rentals (9)
Tuxedos (8)
Invitations (8)
Officiants (8)
Coordinators (7)
Honeymoon (6)
Jewelry (5)
Ceremony Sites (255)
Reception Sites (142)
See All Vendors >
June, 2009 in Elm Grove
- Photographer: A Moment in Time Photography
- Wedding Fashion Vendor: A Savvy Bride
- Ceremony:
- Hotel: Brookfield Suites Hotel
- Reception:
- Hotel: Sheraton Hotel
- Hotel:
A Moment in Time Photography
W157 N9974 Bayberry CircleGermantown, WI 53022, USA
"Rick did a fabulous job! The photos turned out excellent and he was willing to work with me on things we really wanted and price. I would HIGHLY recommend him to any Milwaukee area bride!"
Wedding Fashion Vendor:
A Savvy Bride
17700 West Capitol Drive #5aBrookfield, WI, United States
"BEWARE!! Horrible service, and very unfriendly staff. I waited SIX MONTHS for my bridesmaids dresses which were sitting in their basement for a month of that time! No phone call they were ready! DO NOT GO THERE!"
14700 Watertown Plank Rd
Elm Grove, WI 53122, US
"Community United Methodist Church in Elm Grove, WI Although the cost to reserve the church was a little pricey, it was well worth it to have Paster Sue do our wedding. Many of our guests said it was the best wedding they have ever been too, because of her sermon and personal touchs! The wedding coordinator was also a huge help for me."
1905 N Calhoun Rd
Brookfield, WI 53005
"The Venice Club in Brookfield, WI The food was exceptional! If you reserve the hall on a Friday or Saturday night you have to spend at least $5750 (not including tax & tip) so if you plan to have a smaller wedding this may not be the place to do it. Food & alcohol prices were cheaper compared to other halls I looked at and MUCH better tasting. You do not get to taste their food for free, however they do give you a sizable discount when you come in. I was very happy here!"


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Friday, December 9, 2011
5 comments:
- 84829942-3a88-11e0-83da-000bcdcb5194 said...
Why American men should boycott American women
Boycott American Women
I am an American man, and I have decided to boycott American women. In a nutshell, American women are the most likely to cheat on you, to divorce you, to get fat, to steal half of your money in the divorce courts, don't know how to cook or clean, don't want to have children, etc. Therefore, what intelligent man would want to get involved with American women?
American women are generally immature, selfish, extremely arrogant and self-centered, mentally unstable, irresponsible, and highly unchaste. The behavior of most American women is utterly disgusting, to say the least.
This blog is my attempt to explain why I feel American women are inferior to foreign women (non-American women), and why American men should boycott American women, and date/marry only foreign (non-American) women.
Tens of millions of American men have had their lives completely destroyed by American women through the following crimes:
1. False rape accusations (it has been proven that up to 80 percent of rape accusations are FALSE)
2. False domestic violence (DV) charges (same as above)
3. Financial destruction of men in divorce courts through alimony and support payments (women get up to 95 percent of their ex-husband's income and savings, as well as the house, car, etc)
4. Emotional destruction of men by ex-wives who have stolen their children from them and forbidden contact
5. Divorced dads who commit suicide as a result
Not one single American woman has EVER condemned their fellow American women for committing these crimes against men. Silence means consent. Therefore, American women support and enjoy destroying men's lives and causing men to commit suicide. Apparently, American women think it is okay to be a criminal, just as long as you are a woman. Therefore, is it any surprise that a huge percent of American men no longer want anything to do with American women, other than using them for easy sex and then throwing them away?
Over 50 percent of American women are single, without a boyfriend or husband; so the fact is most American men no longer want to marry American women. Let these worthless American women grow old living alone with their 10 cats.
BRAND NEW: Buy the Boycott American Women book. After reading this book, I promise you that you will never even think about dating an American woman ever again, or else I will give you your money back. That's right, if you are not convinced by my book, I will give you a full refund!
Buy the Boycott American Women eBook now for just $4.99:
Boycott American Women eBook - December 10, 2011 at 3:31 AM
- BP said...
Ooooops, vilken kommentar du fick här. Du är ju egentligen fel person att få den kommentaren men jag fick iaf ett gott skratt;-)
Teckningen är sådär. - December 10, 2011 at 1:33 PM
- craig wilson said...
Is that zombie an American victim?!!
Sheesh, way to ruin the awesome artwork mood. - December 10, 2011 at 11:51 PM
- Eric Z said...
Nice work and great tonal work. and is it just me or does that zombie lady looks a bit "cold"...(winkyface)
- December 12, 2011 at 11:40 AM
- Federico Distefano said...
x BP:
x Craig Wilson:
If you have a gun and use her like a target, yes.
x Eric Z:
Thank you!
:) - December 16, 2011 at 2:38 AM


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Saints know Sunday's game is big for both teams
The New Orleans Saints wrapped up their last day of full on practice Friday. Saturday they?ll put the finishing touches on things prior to boarding a plane and heading up to Charlotte for Sunday?s game against the Panthers.? [ Read more ]
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About Worcester Victorian Christmas Fayre
The Worcester Victorian Fayre is a wonderful Christmas fayre for all the family to enjoy with rides and activities for the children, as well as stalls for you to buy those all important special gifts for loved ones. There's dance displays, choirs and music performances too, really adding to the Christmas atmosphere and of course you can't forget the traditional roast chestnuts and mulled wine.
Places to stay in Worcester for the Worcester Victorian Christmas Fayre
Eventful Stays offers a great range of luxury self catering accommodation in and around Worcester for the Worcester Victorian Christmas Fayre. From apartments to townhouses, we have everything you need to ensure your stay in Worcester is as magical as the event itself.
Do you want to earn extra rental income from your home in Worcester?
If you own a property in Worcester and would like to earn extra income from renting it out, Eventful Stays will list your property free of charge. We specialise in renting high-end luxury accommodation close to major events in the UK and we are looking for more properties in the Worcester area for the Worcester Victorian Christmas Fayre.


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Looking for a Used LX 470 in your area?
CarGurus has 289 nationwide LX 470 listings starting at $4,300.
2007 Lexus LX 470 Overview
The Lexus LX series represented the Japanese luxury automaker's initial trek into the SUV market. First appearing in the mid-1990s as the LX 450, this midsize SUV was virtually indistinguishable from Toyota's Land Cruiser. While this similarity spoke to the LX's off-road ability, many were left with questions about its value in its class. In the past decade, the LX has seen little change, and 2007 is essentially a carry-over year. A Limited Edition LX 470 is new for this year, with production numbers commensurate with its name. The second generation, which began back in 1998, will end after 2007. An all new LX should appear for the 2008 model year.
Aside from the special edition that is offered this year, the LX 470 is only offered in one trim. While the LX is a capable 4x4, its interior has been dressed to be reminiscent of the LS sedan. Standard features include a premium 11-speaker Mark Levinson audio system with an in-dash CD changer, leather/heated/power seats, automatic climate control, a DVD-based navigation system with a backup camera, a moon roof, auto-dimming mirrors, and most other things you could think of. Rear seat DVD entertainment and the Lexus Night View System are optional.
The LX 470 houses a 4.7 liter V-8 inside its rugged body-on-frame construction. The V-8 is enhanced by Toyota's Variable Valve Timing + intelligence technology, which boost engine output while improving fuel economy and lowering emissions. The 5590 lb. LX receives 268 horsepower and 328 pound-feet of torque, which is enough power to pull 6500 additional pounds. The engine is paired with an electronically-controlled five-speed automatic transmission. Drivers can expect gas mileage of 13/17 mpg.
What's your take on the 2007 Lexus LX 470?
Looking for a Used LX 470 in your area?
CarGurus has 289 nationwide LX 470 listings starting at $4,300.
There aren't any questions yet.
- Base
- Avg. Price: $23,145


text: Ninety acres of vinifera grapes grow under the watchful eye of the soaring osprey, our vineyard's namesake. Located on the North Fork of Long Island, our spacious grounds, live music and comfortable tasting room make for an enjoyable visit. Pack a picnic and enjoy our award winning wines. TasteNY Attraction.


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Adopting the Right Dog for You
How to find the right dog for you and your family
Welcome to Petfinder.com! The virtual home of 374,783 adoptable pets from 13,618 adoption groups
Friendliness towards dogs
Friendliness towards other pets
Friendliness towards strangers
Ease of training
Watchdog ability
Protection ability
Cold tolerance
Heat tolerance
The Great Pyrenees is a capable and imposing guardian, devoted to her family and somewhat wary of strangers, human or canine. When not provoked, she is calm, well-mannered and somewhat serious. She is very gentle with her family and children. She has an independent, somewhat stubborn, nature and may try to dominate a less secure handler. Some are not good off leash and may wander away. The Great Pyrenees tends to bark a lot.
The Great Pyrenees needs daily exercise to stay in shape, though her needs are not excessive. A moderate walk will usually suffice. She enjoys hiking, especially in cold weather and snow. She does not do well in hot weather. This breed enjoys being with her family indoors. Her coat needs brushing once or twice weekly, daily when shedding. She may drool at times, and she tends to be a messy drinker.
Major concerns: CHD, patellar luxation
Minor concerns: entropion, OCD, skin problems, osteosarcoma
Occasionally seen: ChD, gastric torsion, otitis externa, panosteitis
Suggested tests: hip, knee, (eye)
Life span: 10-12 years
Bernese Mountain Dog
Doberman Pinscher
Great Dane
Great Pyrenees
Greater Swiss Mountain Dog
Some animal welfare organizations with Great Pyreneess ready for adoption:
How to find the right dog for you and your family
Why adopting an older dog has many advantages
Prepare in advance to make sure your home is safe for your new pup


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more... - From the President
Lena Horne: In Her Own Voice, An American Masters Special on WXXI-TV
Lena Horne: In Her Own Voice, An American Masters Special on WXXI-TV
Sun, 05/16/2010 - 7:00pm
In Memory of: Lena Horne 1917-2010.
This special encore presentation of Lena Horne: In Her Own Voice, An American Masters Special airs Sunday, May 16 at 7 p.m. on WXXI-TV (DT21.1/cable 1011 and 11).
Lena Horne tells the story of her journey to reluctant stardom. In addition to exclusive interviews with Horne and her daughter, the documentary, directed by Susan Lacy, includes interviews with Ossie Davis, Alan King, Liz Smith and others who worked with the performer. Clips from signature movies like "Stormy Weather" and MGM films of the 1940's are interwoven with her early TV appearances to accentuate her life story. Lena Horne died in May 2010, at the age of 92.


text: Serowe Art Gallery "To the Water" Size: 980 mm X 730 mm, Framed Original Oil on Canvas by South African Wildlife Artist Dane Willers Auctioned as an Unframed but Stretched Canvas Gallery Prtice: R14500.00, Framed Postage: R280.00 within RSA Do look at ALL our ratings!! Certificate of Authenticity/Valuation supplied to full value. These works are done on high grade Canvas, stretched on a stretcher frame and not done on some board, or canvas covered board. Only profesisonal Winton & Newton Oil paints are used. See all our items at: http://www.bidorbuy.co.za/seller/951291/Serowe_Art_Studio PLACE YOUR BEST BID EARLY - WILLING TO CLOSE THIS AUCTION ON THE FIRST BID. |


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Recent Reviews
We stayed at this villa for 6 days and thoroughly enjoyed it! there were only 5 of us but it would have comfortably housed 10-12. The villa and facilities inside are perfect for big families/groups. Outdoor area with kitchen is ideal. We did not hire a car and managed to get around using taxi's/ local bus' (although sometimes unreliable).
A welcome pack with info on the area (such as map to nearest supermarket etc) and household may have been helpful as we didn't meet the owner until the 3rd day. The estate where the villa is situated is very quiet and there is not much going on, apart from tennis courts and one bar. However, the local city, Alcala de Henares is only a 10 minute cab journey away. The city is beautiful and has lots of great bars and restaurants. We would have loved to use the bikes in the villa, however many were unusable unfortunately.
We really loved the villa and would definitely recommend for all groups. Frederic is very helpful and easily contactable. Thanks again!


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In this article I will explain what a pixel is and for what reasons it can get stuck. I will then show you how you can identify bad pixels on your LCD screen and what you can do to possibly fix them yourself. Only after you have tried all your options, is it time to claim your warranty.
What Are Pixels?
Simply put, pixels are single dots in a raster image and all of them put together make up the entire image.
What you are seeing on your computer screen right now, technically, is a raster image made up of hundreds of pixels. On CRT, LCD, LED, or plasma displays, however, pixels are not static, they display moving and ever changing images, therefore they must change colors. To achieve this, every pixel is divided into three sub-pixels, i.e. red, green, and blue. Each sub-pixel is powered by a transistor that controls the on/off state of the pixel.
What Is A Bad Pixel?
A bad pixel is a pixel that does not display the color you expect it to display. It is either stuck in a certain color, temporarily off, or permanently off, which equals a dead pixel.
Generally, what can happen is that the liquid crystal inside a sub-pixel is not evenly distributed, therefore that pixel never lights up. If all three pixels are affected, you get a pixel that is temporarily off. Another scenario is that a transistor is broken and consequently the pixel is permanently off (dead) or possibly also permanently on (burning). The latter is the worst case scenario as you cannot fix a broken transistor. You may, however, be able to fix problems with the liquid crystal.
How Can I Find Out Whether Or Not I Have Bad Pixels?
There are several online tools and applications you can run on your computer to identify bad pixels.
I have previously recommended 5 Ways To Fix A Stuck Pixel On Your Screen 5 Ways To Fix A Stuck Pixel On Your Screen Read More the online LCD test tool from Flexcode. Since then, they have provided an updated version of the tool, which comes with a much better interface, making it easier to use. Simply run all five color tests (green, blue, red, white, and black) one after the other and check your screen for irregularities.
Like the previous version, this one also includes a 'massage the pixel' option, which may possibly fix a stuck pixel by over-stimulating it. So should you spot bad pixels or find darker regions on your LCD screen, run the 'Fix My Screen' mode for a longer period of time (about one hour) to revive the affected pixel/s.
IsMyLcdOK is a portable application for Windows. You can easily zap through the various test screens using number keys, as well as F2 through F5. F1 brings up the options overview.
Mac users may want to try Pixel Fix, a Dashboard widget to cure stuck pixels on LCD screens.
I Found a Bad Pixel – How Can I Fix It?
The only way to fix a stuck pixel is to over-stimulate it and pray that it comes to life again. You can over-stimulate pixels in two different ways – you can either send rapidly changing signals to the respective pixel or you can manually apply pressure to the respective area on your screen.
I have described both methods in my previous article 5 Ways To Fix A Stuck Pixel On Your Screen 5 Ways To Fix A Stuck Pixel On Your Screen 5 Ways To Fix A Stuck Pixel On Your Screen Read More . Briefly, you can use the 'Fix My Screen' option of LCD2 (see above) to treat the entire screen. If you can't have your whole screen blocked for up to an hour, you can also use the 'undead pixel' option of UDPixel (screenshot below), to locally fix a bad pixel. Finally, the most successful method appears to be the manual one, i.e. applying pressure to the area of your screen that has the dead pixel.
For a thorough description, please refer to my previous article 5 Ways To Fix A Stuck Pixel On Your Screen 5 Ways To Fix A Stuck Pixel On Your Screen Read More .
If none of the methods worked instantly, try again and try several times. As you can see from the comments to the first article, it is very possible to fix bad pixels!
For more information about LCD monitors and a huge selection of test images, check out The Lagom LCD monitor test pages.
Have you ever succeeded in fixing a stuck pixel and how did you do it?
Image credits: Luis Azevedo, photobank.kiev.ua
Leave a Reply
At the end of the day, dead / Light pixels should not affect the actual experience with a computer, whether you are a graphic enthusiast or not. Most likely personal OCD rather than the actual problem it might have.
Having used computer for 20 years and never care about dead light pixels and everything is fine.
I have a rotating picture box on the homepage of my website. One of the images in the rotating picture box has what seems to be white/blue "stuck" pixels. 1 second before the image fades into the next image, they disappear. These "stuck" pixels are not on anything else but this one image on the website (this same image does not contain these spots in the tiff file or jpeg file). Do you know what I can do to remove them or what is causing them?
Do you see these 'stuck pixels' only when you view this picture on your homepage on one specific computer or do you also see the weird pixels on any other monitor?
If you are worried that this is a fault of your monitor, I recommend running a test tool like the one introduced in the article above. If you find that there really are 'stuck pixels', try the fixes suggested in the article.
If, however, this is a fault with the image displayed on your homepage and not with your monitor, then we have a completely different issue and I recommend you to ask your question on MakeUseOf Answers.
I'll test my LCD to find the Bad pixels on it


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I will probably have more to say tomorrow, when I'm not so ecstatic and dead.
Looks like we've got our first 20-goal scorer in the NHL this season. (;
Suck it, Pens. It's sad how much I liked you two years ago, and the love is slowly dwindling away...I don't think I'll ever be able to wear my Crosby jersey again. What a shame.
This was a great effort by the team tonight, and I'm glad they rallied back to beat the Penguins. Another heartbreaking loss to them.
Let's go, Buff-a-lo!
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237 of 2223
This patient wanted to get rid of the "waddle" under his neck. he was not interested in performing a lower face and neck lift, and opted for doing a neck lift only, which focused solely on the neck skin and not the jowls or jawline.
By uploading this photo, Richard Balikian, M.D. has agreed with RealSelf.com policy that they've secured proper patient consent.


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Shakespeare's "problem play" Measure for Measure is staged infrequently, yet is almost eerily contemporary in its subject. For the production on the Goodman Theatre main stage, Artistic Director Robert Falls finds placing the action in a declining, yet over-the-top 1970s Manhattan
Archive for April 1, 2013
Bill McGlaughlin Profiles Gabriel Fauré
Monday, April 1, 2013 by Noel Morris
Student of Camille Saint-Saens and teacher to Maurice Ravel, Gabriel Fauré lived and helped drive one of the most fascinating eras in music. He was choir master at Saint-Sulpice when Widor was organist, and part of an association which included Bizet, Franck, Lalo and Massenet


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Don't forget your passport
When traveling to Mexico, it is best to remember a passport. Nick does not. When he and Jess head south for a romantic vacation in the Season 3 premiere, problems follow (as reported by TVLine). Nick's lack of documentation means that Schmidt and Winston have to come to the rescue.
That rescue goes about as well as you would expect for a rescue from Schmidt and Winston. It seems that all of this results in many encounters with a tazer and a rather aggressive security guard.
Sibling rivalry — will there be a real sibling?
Zooey Deschanel of "New Girl" and Emily Deschanel of "Bones" are real sisters starring in shows on the same network. But is there a chance that they could play sisters on "New Girl"?
Casting the busy Emily Deschanel (who is the mother of a toddler, in addition to her lead role on a drama series) may not be likely, but there will be a sister for Jess Day in Season 3. TVGuide.com reports that the sister will arrive at some point this year. Series creator Liz Meriwether teases the character as being "trouble" and "fun."
Let's all just pray for some double-Deschanel fun in the process!


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I need help with this assignment
Follow the bullet points of P2 {task 2} Link to your topic anti smoking!
Hello everyone I need this assignment in 4 hours you have to follow all the instructions that are written! no plagiarism cause I have a program that can see if the assignment is plagiarized or not! you have to do only M1. Put only the price that I put otherwise if the offers will be more high will not be considered. you must to do it on a word document , there is no limit of words but it would be better if you can write in 500 words minimum. Follow the bullet points of P2 {task 2} you have to put also the reference!!!
instructions: IMG_0013.JPG
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