(Stroke. 1995;26:864-869.)
© 1995 American Heart Association, Inc.
Articles |
Nitro-L-Arginine Analogues
Dose- and Time-Related Nitric Oxide Synthase Inhibition in Brain
From the Departments of Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Md.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Background and Purpose The purpose of the present study was to measure cortical nitric oxide synthase (NOS) activity and determine the appropriate doses of N
-nitro-L-arginine methyl ester
(L-NAME) or N-nitro-L-arginine
(L-NNA) for near-complete enzyme inhibition in dogs, cats, and pigs. We
anticipated that NOS inhibition was dose- and time-dependent and
questioned if the dose-response relationship was related to the
specific drug or animal species. Methods Saline or L-NAME or L-NNA in escalating doses was administered to pentobarbital-anesthetized pigs, dogs, and cats. Brain temperature and arterial blood gas, hemoglobin, and blood pressure levels were maintained within the physiological range. Cortical tissue was biopsied at baseline and 30, 120, and 360 minutes after agent administration for measurement of NOS activity by isotopic assay of the conversion of [14C]arginine to [14C]citrulline.
Results L-NAME produced >70% enzyme inhibition at a dose of 20 mg/kg across the species tested. Arterial blood pressure was elevated at 30 minutes after L-NAME treatment. However, consistent decreases in brain NOS activity required a longer period of time. Near-complete inhibition was apparent in most animals by 120 minutes and persisted for 6 hours after administration. A smaller dose of L-NNA was required for >70% enzyme inhibition in the cats and dogs (10 mg/kg). Near-complete NOS inhibition was evident in most animals at 30 minutes after L-NNA administration, which also persisted for 6 hours. In pigs, this same level of inhibition required 20 mg/kg.
Conclusions These results suggest that administration of L-NAME and L-NNA diminishes brain NOS activity in a dose- and time-dependent manner and that the duration of effect is at least 6 hours.
Key Words: cerebral circulation • nitric oxide • nitric oxide synthase • nitro-L-arginine analogues
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nitric oxide (NO) or an NO-containing compound plays an instrumental role in cerebral vascular smooth muscle reactivity1 and contributes to basal cerebral blood flow.2 3 4 NO is synthesized via the conversion of the amino acid L-arginine to L-citrulline by the enzyme NO synthase (NOS).5 Various NOS isoforms have been localized in vascular endothelium,6 neurons,7 8 and astrocytes9 within the brain. We3 4 10 11 12 13 14 15 16 and others17 18 19 have used a class of competitive NOS inhibitors, the nitro-L-arginine analogues, as a means of investigating the role of NO in cerebral vasodilatory mechanisms. These agents have been administered in numerous species, including mice,17 rats,18 rabbits,20 piglets12 and pigs,15 cats,3 10 14 dogs,4 and monkeys.16 One of the most widely used agents is N
-nitro-L-arginine methyl ester
(L-NAME). L-NAME, like other alkyl esters of arginine, is a potent
competitive inhibitor of NOS but also may have concentration-dependent,
muscarinic-receptor antagonist properties.21 Because of
the potential nonspecificity of L-NAME, other agents such as
N-nitro-L-arginine
(L-NNA)19 21 are increasingly used in NO-related
investigations. Both L-NAME and L-NNA have been administered in many experimental paradigms at varying doses,
over different time courses, and in different species. These paradigms
frequently have been performed without concurrent documentation of
appropriate enzyme inhibition. The purpose of the present study was to measure in vitro cortical NOS activity and determine the appropriate doses of L-NAME and L-NNA for near-complete enzyme inhibition in dogs, cats, and pigs. In rats L-NAME administered at 20 mg/kg IV resulted in a stable level of inhibition (52±3%) within 2 hours.22 We anticipated that NOS inhibition was dose- and time-dependent and questioned if the dose-response relationship was related to the specific drug or animal species.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study was approved by the Johns Hopkins Animal Care and Use Committee and is in compliance with the guidelines of the National Institutes of Health for care and handling of animals. Three species of both sexes were used: 3- to 4-month-old mixed breed pigs (15 to 20 kg, n=21) and mongrel cats (3 to 4 kg, n=22) and dogs (20 to 25 kg, n=34). Anesthesia was induced with pentobarbital by intraperitoneal injection in pigs and cats (65 to 100 mg/kg) or intravenous injection in dogs (15 to 20 mg/kg). Additional intravenous pentobarbital was administered during surgery and the experimental protocol if the animal moved in response to stimuli. Orotracheal intubation (cats and dogs) or tracheostomy (pigs) was performed, with subsequent mechanical ventilation to maintain constant end-tidal CO2 and arterial blood gases. Supplemental oxygen was administered to maintain arterial PO2 at >150 mm Hg. Both femoral veins were cannulated for administration of fluids and drugs, and an aortic catheter was inserted through the femoral artery for monitoring mean arterial blood pressure (MABP). In control animals, a balloon-tipped catheter (Fogarty arterial embolectomy catheter, Baxter Healthcare Corporation) was placed in the descending aorta at the level of the renal arteries (4F in cats, 7F in pigs and dogs). The balloon catheter was inflated in these animals during the protocol to elevate MABP to levels comparable to those of drug-treated animals. The temporalis muscle was retracted bilaterally, and four craniectomy sites (2 cm in diameter) were drilled in the skull for subsequent cortical tissue sampling. At each site, the dura and arachnoid remained intact until the biopsy was obtained. A thermistor (Mon-A-Therm, LaBarge) was placed in the cranial epidural space through one craniotomy site. Epidural temperature was maintained at 38±1°C with the use of a heat lamp and heating pad.
MABP was continuously measured with Statham P-23 pressure transducers, referenced to the left atrium, and recorded on a Gould-Brush recorder. Arterial PO2, PCO2, and pH levels were measured with a Radiometer BMS-3 system (ABL 3). Arterial oxygen content, saturation, and hemoglobin concentration were determined with a Radiometer Hemoximeter (model OSM3). Arterial glucose concentration was measured with a YSI model 2300 Stat Glucose/Lactate Analyzer and maintained with 5% dextrose/0.45% saline as needed. Intravenous sodium bicarbonate was also administered as needed to regulate systemic pH at 7.30 to 7.45.
After a 30-minute stabilization period, the dura was gently lifted, and four tissue samples (approximately 50 mg each) were cut with a number 11 scalpel blade from the brain at sequential time points in a clockwise manner such that the first biopsy site was contralateral to the last site. One cortical sample was obtained per time point, and each craniectomy served as the site of one sample. Bleeding at these sites was controlled with avian collagen. After the baseline biopsy was obtained, each animal was treated with either saline (10 mL/kg), a specific dose of L-NNA (Sigma Chemical Company; in 10 mL/kg warmed stirred sterile water), or a specific dose of L-NAME (Sigma; in 10 mL/kg room-temperature sterile saline) administered by intravenous infusion over 10 minutes. Doses were determined in a post hoc fashion depending on the results of ongoing experiments. The doses that were tested were different for the two blockers because of the different dose responses at the low-dose (<5 mg/kg) and high-dose (>30 mg/kg) range. The following doses were tested: L-NNA at 1, 5, 10, 20, 30, and 50 mg/kg and L-NAME at 10, 20, 30, 40, and 50 mg/kg. Subsequent brain biopsies were then obtained from each of the three other craniotomy sites at 30, 120, and 360 minutes after infusion. The final biopsy was obtained from the thermistor-instrumented craniotomy. At the end of each experiment, the animal was killed with an intravenous injection of potassium chloride.
Once collected, the sample was rinsed in ice-cold buffer (50 mmol/L Tris HCl, pH 7.4, with 2 mmol/L EDTA), then sonicated and centrifuged at 10 000g for 15 minutes at 4°C. The supernatant was frozen and then thawed for analysis 1 to 5 days later. NOS activity was assayed in triplicate using a modification of the technique described by Bredt et al.3 10 23 In this technique, the conversion of [14C]L-arginine to [14C]L-citrulline is determined as an indirect measure of NOS activity. In brief, the reaction is initiated (20°C) by adding 25 µL of the supernatant fraction to 100 µL of reaction mixture (1 µmol/L [14C]L-arginine, 1 mmol/L NADPH, and 1 mmol/L CaCl2). The calcium dependence of this reaction was established previously.24 The reaction is terminated after 30 minutes by adding 2 mL of stop buffer (30 mmol/L HEPES, pH 5.2, and 3 mmol/L EDTA). A time of 30 minutes was chosen because in vitro reaction velocity is constant over this period, as has been previously reported.25 [14C]L-Citrulline is eluted on chromatography columns (resin AG-50WX8, Na+ form, pH 7.0) and quantified by liquid scintillation spectroscopy (Beckman LS 1800). The total citrulline recovery was calculated from specific activity of the [14C]L-arginine, correcting for counting efficiency. Parallel samples were processed in the presence of 100 µmol/L L-NAME, demonstrating greater than 99% inhibition of L-arginine conversion as evidence that all citrulline production was consequent to NOS activity. Protein concentration was measured by the method of Bradford,26 and the data are expressed as picomoles per minute per milligram of protein.
The number of animals treated with each dose of the inhibitors was minimized; for example, when near-complete inhibition was consistently observed, no more animals were tested at that dose. Consequently, the number of animals in the various dose/drug groups was small and is presented as pooled data in some instances. Baseline physiological variables were pooled by species. MABP was pooled by species at four time points and then analyzed with a one-way ANOVA and Dunnett's test to determine whether there were drug-induced differences in MABP compared with baseline values. Statistical significance was declared if P<.05. The dose-response for each inhibitor is presented as individual animal responses and was not analyzed statistically. Since variability in interanimal NOS activity was large, data are expressed as percentages of individual baseline values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Arterial blood gas, glucose, and hemoglobin levels were maintained within physiological ranges throughout the protocol in all animals. Table 1
presents baseline physiological variables
for the pooled species. MABP values by species for animals receiving
either saline, L-NAME (all doses), or L-NNA (all doses) are shown in
Table 2. As expected, L-NAME treatment increased MABP in
all species. Treatment with L-NNA also produced increases in MABP.
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NOS activity was expressed as a rate at 30 minutes into the reaction.
Baseline activity of NOS was 4.20±1.25, 4.21±0.48, 6.43±0.92
pmol/min per milligram of protein for cats, dogs, and pigs,
respectively. There were no differences between these groups. The
interspecies and interanimal variability in control animals was large,
and NOS activity ranged from 30% to 150% of baseline values. NOS
activity over time is shown in Fig 1 for saline-treated
cats (n=7), dogs (n=7), and pigs (n=6). The lowest activity observed in
this control group was 30% of baseline; therefore, we evaluated
effective NOS inhibition with L-NAME or L-NNA (subsequent dose-response
plots) as the proportion of animals with >70% NOS inhibition.
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, n=7), dogs (
,
n=7), and pigs (
, n=6). Means±SEM are presented adjacent to
these time points. Because all data are presented as a percentage
of baseline, no baseline values (which by definition equal 100%) are
presented.
Fig 2 displays NOS activity in individual animals
treated with the various doses of L-NAME. Each panel summarizes the
percentage of NOS activity at the three time points tested (30, 120,
and 360 minutes after administration). Enzyme activity with doses 
10
mg/kg L-NAME was largely reduced in many, but not all, animals by 30
minutes after infusion (Fig 2, top). Larger doses (20 mg/kg) produced
70% inhibition in all but 4 (3 dogs, 1 cat) of the 21 treated animals.
By 120 minutes (Fig 2, middle), consistent 70% inhibition with a dose
of 20 mg/kg was evident in all but 1 dog. By 360 minutes (Fig 2,
bottom), 70% inhibition was present in all animals receiving doses
of 20 mg/kg. Cats, unlike dogs or pigs, exhibited near-complete
inhibition at this time point with 20 mg/kg doses.
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Fig 3 plots the L-NNA dose-response in treated animals
at the same three time points. At 30 minutes, 10 mg/kg L-NNA produced
70% NOS inhibition in cats and dogs but not in pigs, which required 20
mg/kg. At 120 minutes, near-complete inhibition was evident in dogs at
doses of 10 mg/kg, with 70% inhibition in cats. With doses of 20
mg/kg, inhibition was near complete in all but 2 (1 pig, 1 cat) of the
15 treated animals. At 360 minutes, 70% inhibition was present in
all but 1 pig with 10 mg/kg and in all 15 animals with 20 mg/kg. The
signal-to-noise ratio, calculated by disintegrations per minute of
signal divided by disintegrations per minute of background in
pigs, dogs, and cats with NOS inhibition >70%, was low
(1.7±0.7, 1.2±0.3, and 1.1±0.3, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study demonstrates that intravenous administration of the nitro-L-arginine analogues L-NAME and L-NNA yields a dose- and time-related diminution of brain NOS activity. L-NAME produced >70% enzyme inhibition at a dose of 20 mg/kg across the species tested (cats, dogs, and pigs). MABP was elevated at 30 minutes after L-NAME treatment; however, consistent depression of brain NOS activity required more time. Near-complete inhibition was apparent in most animals by 120 minutes, which persisted for 6 hours after administration. A smaller dose of L-NNA was required for >70% enzyme inhibition in cats and dogs (10 mg/kg). Near-complete NOS inhibition was evident in most animals at 30 minutes after L-NNA administration, which also persisted for 6 hours. In pigs, this same level of inhibition required 20 mg/kg.
The dosages of L-NNA required for >70% inhibition were similar in cats and dogs but appeared to be larger in pigs. This observation may be explained by the greater apparent lipophilicity of L-NNA, as indicated by its relative insolubility in an aqueous solution compared with L-NAME. The initial volume of drug distribution of such lipid-soluble drugs would be larger in pigs because of the high percentage of body fat relative to cats or dogs. Consequently, larger doses of L-NNA may be required to assure adequate drug availability to the brain in pigs. The extent and temporal pattern of NOS inhibition after administration of L-NAME are in agreement with data described in rats.22 L-NAME, when administered to rats (10 mg/kg), resulted in a stable level of inhibition (40±5%) within 2 hours. There was no additional inhibition produced with doses >20 mg/kg. Two hours were required to reach a stable level of inhibition because L-NAME relies on diffusion to enter the cytosol. Unlike L-NAME, L-arginine and other arginine analogues interact with the y+ transporter that facilitates entry into the cytosol.27
Both L-NNA and L-NAME are nonselective inhibitors of NOS and presumably act on both constitutive and inducible isoforms of the enzyme. The present study was designed to determine the dose required for near-complete enzyme inhibition and did not address the specific isoform target for inhibition. Given the significant inhibition observed within 30 minutes of administration, it is unlikely that inducible NOS from macrophages was the major target for L-NAME and L-NNA. The immediate, systemic hypertensive effect of these agents suggests that vascular endothelial NOS was inhibited. However, our sampling technique accessed predominantly gray matter in the parietal and temporal cortical areas. Since neurons and glia are estimated to constitute the majority of cortex brain volume,28 the major fraction of NOS measured in our samples most likely originates in neurons and glia rather than endothelium. Furthermore, anesthetic requirements decrease after L-NAME administration,29 suggesting that this agent crosses the blood-brain barrier and inhibits neuronal NOS.
We used a well-established isotopic conversion assay for evaluating cytosolic NOS activity.10 23 Our results indicate that basal enzyme activity is surprisingly variable over time in anesthetized large animals during steady-state conditions. This variability may reflect regional cortical differences in NOS activity that were not fully minimized by our sampling protocol. The results with systemic inhibitors must also be interpreted with a further methodological consideration in mind: the in vitro assay indirectly reflects in vivo enzyme activity. This concern is important if, for example, the drug is unable to reach the site(s) of brain NOS in vivo but, after sonication and disruption of the tissue, may have access to the enzyme. Under such circumstances, the assay may overestimate the percentage of inhibition achieved for the doses administered. Other sources of variability in the assay include the variable period of supernatant freezing, heterogeneity of the [14C]arginine ligand, and inhomogeneity of the Dowex column. Some of these problems were addressed by constantly monitoring the background activity, which, when it exceeded 300 cpm, was replaced with new Dowex and new labeled arginine. Quality checks of the Dowex assured a 2% recovery of arginine and a 90% to 99% recovery of citrulline. It is not known whether any species differences exist regarding substrate or cofactor requirements for this enzyme reaction.
A wide range of L-NAME and L-NNA doses have been used in numerous investigations that center on NO as a mediator of cerebral vasodilatory responses during hypoxia, hypercapnia, and functional activation, as well as a potential neurotoxin in cerebral ischemia. Despite the number of investigations in these areas, considerable controversy remains regarding NO and its actions in the brain. Some of the difficulty in precisely evaluating the importance of NO in cerebrovascular regulation relates to the range of inhibitory agents, doses, and administration time frames used. This is complicated by the lack of documentation of acceptable inhibition and residual NOS activity. Others have emphasized the importance of establishing appropriate enzyme inhibition in experimental protocols that administer L-arginine analogues.30 The present study demonstrates that substantial doses of either L-NAME (20 mg/kg) or L-NNA (10 mg/kg in cats and dogs, 20 mg/kg in pigs) are required to consistently inhibit brain NOS activity by >70%. Smaller doses do not produce adequate inhibition in these species. In addition, 30 minutes is not sufficient for achieving near-complete enzyme inhibition when L-NAME is used.
We conclude that the dose-response relationships are not greatly dissimilar for L-NAME and L-NNA in anesthetized animals. Apparent species differences among cats and dogs versus pigs likely are based on volume of drug distribution. Given the considerable interanimal variability in response to systemic inhibition, measurement of NOS activity and documentation of adequate inhibition as part of each experimental protocol should be considered.
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Acknowledgments |
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This work was supported by US Public Health Service National Institutes of Health grant NS-20020. We wish to thank Jeffrey R. Kirsch, MD, Joseph R. Tobin, MD, and Linda Gorman, PhD, for their kind and critical review of this manuscript.
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Footnotes |
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Reprint requests to Richard J. Traystman, PhD, Department of Anesthesiology/Critical Care Medicine, The Johns Hopkins Medical Institutions, Blalock 1408, 600 N Wolfe St, Baltimore, MD 21287-4963.
Received August 8, 1994; revision received December 1, 1994; accepted February 20, 1995.
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S. S. Malik and J. E. Fewell Thermoregulation in rats during early postnatal maturation: importance of nitric oxide Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2003; 285(6): R1366 - R1372. [Abstract] [Full Text] [PDF]
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T. Nagaoka, T. Sakamoto, F. Mori, E. Sato, and A. Yoshida The Effect of Nitric Oxide on Retinal Blood Flow during Hypoxia in Cats Invest. Ophthalmol. Vis. Sci., September 1, 2002; 43(9): 3037 - 3044. [Abstract] [Full Text] [PDF]
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H. Xu, G. D. Fink, and J. J. Galligan Nitric oxide-independent effects of tempol on sympathetic nerve activity and blood pressure in DOCA-salt rats Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H885 - H892. [Abstract] [Full Text] [PDF]
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A. A. Steiner, J. Antunes-Rodrigues, S. M. McCann, and L. G. S. Branco Antipyretic role of the NO-cGMP pathway in the anteroventral preoptic region of the rat brain Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2002; 282(2): R584 - R593. [Abstract] [Full Text] [PDF]
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H. Xu, G. D. Fink, A. Chen, S. Watts, and J. J. Galligan Nitric oxide-independent effects of tempol on sympathetic nerve activity and blood pressure in normotensive rats Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H975 - H980. [Abstract] [Full Text] [PDF]
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A. Sener and F. G. Smith Nitric oxide modulates arterial baroreflex control of heart rate in conscious lambs in an age-dependent manner Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2255 - H2263. [Abstract] [Full Text] [PDF]
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A. A. Steiner, E. C. Carnio, and L. G. S. Branco Role of neuronal nitric oxide synthase in hypoxia-induced anapyrexia in rats J Appl Physiol, September 1, 2000; 89(3): 1131 - 1136. [Abstract] [Full Text] [PDF]
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M. C. Almeida, E. C. Carnio, and L. G. S. Branco Role of nitric oxide in hypoxia inhibition of fever J Appl Physiol, December 1, 1999; 87(6): 2186 - 2190. [Abstract] [Full Text] [PDF]
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M. A. El-Haddad, C. R. Chao, S.-X. Ma, and M. G. Ross Nitric oxide modulates spontaneous swallowing behavior in near-term ovine fetus Am J Physiol Regulatory Integrative Comp Physiol, October 1, 1999; 277(4): R981 - R986. [Abstract] [Full Text] [PDF]
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M. Matsumoto, Y. Iida, H. Wakamatsu, K. Ohtake, K. Nakakimura, L. Xiong, and T. Sakabe The Effects of NG-Nitro-L-Arginine-Methyl Ester on Neurologic and Histopathologic Outcome After Transient Spinal Cord Ischemia in Rabbits Anesth. Analg., September 1, 1999; 89(3): 696 - 696. [Abstract] [Full Text] [PDF]
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M. Sander, B. Chavoshan, and R. G. Victor A Large Blood Pressure–Raising Effect of Nitric Oxide Synthase Inhibition in Humans Hypertension, April 1, 1999; 33(4): 937 - 942. [Abstract] [Full Text] [PDF]
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C. Baylis, J. Harvey, B. R. Santmyire, and K. Engels Pressor and Renal Vasoconstrictor Responses to Acute Systemic Nitric Oxide Synthesis Inhibition Are Independent of the Sympathetic Nervous System and Angiotensin II J. Pharmacol. Exp. Ther., February 1, 1999; 288(2): 693 - 698. [Abstract] [Full Text]
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R. C. H. Barros and L. G. S. Branco Effect of nitric oxide synthase inhibition on hypercapnia-induced hypothermia and hyperventilation J Appl Physiol, September 1, 1998; 85(3): 967 - 972. [Abstract] [Full Text] [PDF]
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R. Stingele, D. A. Wilson, R. J. Traystman, and D. F. Hanley Tyrosine confounds oxidative electrochemical detection of nitric oxide Am J Physiol Heart Circ Physiol, May 1, 1998; 274(5): H1698 - H1704. [Abstract] [Full Text] [PDF]
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C. L. Schleien, J. W. Kuluz, and B. Gelman Hemodynamic effects of nitric oxide synthase inhibition before and after cardiac arrest in infant piglets Am J Physiol Heart Circ Physiol, April 1, 1998; 274(4): H1378 - H1385. [Abstract] [Full Text] [PDF]
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H. Murakami, J.-L. Liu, H. Yoneyama, Y. Nishida, K. Okada, H. Kosaka, H. Morita, and I. H. Zucker Blockade of neuronal nitric oxide synthase alters the baroreflex control of heart rate in the rabbit Am J Physiol Regulatory Integrative Comp Physiol, January 1, 1998; 274(1): R181 - R186. [Abstract] [Full Text] [PDF]
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B. P. Wagner, R. Stingele, M. A. Williams, D. A. Wilson, R. J. Traystman, and D. F. Hanley NO contributes to neurohypophysial but not other regional cerebral fluorocarbon-induced hyperemia in cats Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1994 - H2000. [Abstract] [Full Text] [PDF]
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R. Bolli, Z. A. Bhatti, X.-L. Tang, Y. Qiu, Q. Zhang, Y. Guo, and A. K. Jadoon Evidence That Late Preconditioning Against Myocardial Stunning in Conscious Rabbits Is Triggered by the Generation of Nitric Oxide Circ. Res., July 19, 1997; 81(1): 42 - 52. [Abstract] [Full Text]
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M. Sander, J. Hansen, and R. G. Victor The Sympathetic Nervous System Is Involved in the Maintenance but Not Initiation of the Hypertension Induced by N{omega}-Nitro-L-Arginine Methyl Ester Hypertension, July 1, 1997; 30(1): 64 - 70. [Abstract] [Full Text]
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C. S. Tietjen, J. R. Kirsch, N. Clavier, and R. J. Traystman Time-Dependent Inhibition of Oxotremorine-Induced Cerebral Hyperemia by N{omega}-Nitro-L-Arginine in Cats Stroke, November 1, 1995; 26(11): 2160 - 2165. [Abstract] [Full Text]
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