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Fetal Sheep (fetal + sheep)

Distribution by Scientific Domains

Life Sciences	47%
Medical Sciences	41%

Selected Abstracts

Effect of Maternal Nutrient Restriction in Early Gestation on Responses of the Hypothalamic-Pituitary-Adrenal Axis to Acute Isocapnic Hypoxaemia in Late Gestation Fetal Sheep

EXPERIMENTAL PHYSIOLOGY, Issue 1 2000
Paul Hawkins
Epidemiological and experimental evidence suggests that maternal undernutrition during pregnancy may alter development of fetal organ systems. We have demonstrated previously that fetal hypothalamic-pituitary-adrenal (HPA) axis responses to exogenous corticotropin-releasing hormone (CRH) + arginine vasopressin (AVP), or adrenocorticotrophin hormone (ACTH), are reduced in fetuses of mildly undernourished ewes. To examine these effects further we tested HPA axis responses to acute isocapnic hypoxaemia in fetal sheep at 114-129 days gestation (dGA), following 15% reduction in maternal nutritional intake between 0 and 70 dGA. Fetuses from control (C) and nutrient-restricted (R) ewes were chronically catheterised and plasma ACTH and cortisol responses were determined at 114-115, 120-123 and 126-129 dGA during hypoxaemia (1 h) induced by lowering the maternal inspired O2 fraction (FI,O2). Basal plasma cortisol concentrations and HPA axis responses at 114-115 and 120-123 dGA did not differ between C and R fetuses. At 126-129 dGA, both plasma ACTH (P < 0.01) and cortisol (P < 0.05) responses were smaller in R fetuses compared to C fetuses. Fetal blood gas status, fetal body weight, body proportions and organ weights did not differ between the groups. We conclude that mild maternal undernutrition alters development of the fetal HPA axis producing a reduction in pituitary and adrenal responsiveness to endogenous stimuli. [source]

Effects of Betamethasone Treatment on Central Myelination in Fetal Sheep: An Electron Microscopical Study

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2 2008
C. Raschke
Summary The long-term effect of betamethasone on the myelination of commissural and associational fibres was investigated in fetal sheep. We studied the corpus callosum and subcortical white matter by electron microscopy. Axons were subdivided into classes according to their axonal diameter: class I: ,0.65 ,m; class II: 0.66,0.84 ,m; class III: ,0.85 ,m. Under control conditions, the different functions of the white matter tracts examined were reflected by three morphological criteria: (1) there was a diverse percentage of axonal classes in the investigated areas. In corpus callosum the axons of class II predominate (47.1%). In the subcortical white matter, class I axons with small diameter are in majority (40.8%). (2) In the subcortical white matter more axons are present, with especially large diameter and hence of axonal class III. (3) The axons of subcortical white matter have thicker myelin sheaths than those of the corpus callosum. Betamethasone administration caused a significant decrease of class II axons in the corpus callosum (36.9%). In corpus callosum, axons of all classes present thicker myelin sheaths. Betamethasone administration resulted in a change in the formation of the myelin sheath in the commissural fibres of the corpus callosum but not in the associational fibres of the subcortical white matter. This could be the morphological correlate to behavioral and cognitive changes known to occur in humans after prenatal glucocorticoid treatment. [source]

Models of white matter injury: Comparison of infectious, hypoxic-ischemic, and excitotoxic insults

DEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 1 2002
Henrik Hagberg
Abstract White matter damage (WMD) in preterm neonates is strongly associated with adverse outcome. The etiology of white matter injury is not known but clinical data suggest that ischemia-reperfusion and/or infection-inflammation are important factors. Furthermore, antenatal infection seems to be an important risk factor for brain injury in term infants. In order to explore the pathophysiological mechanisms of WMD and to better understand how infectious agents may affect the vulnerability of the immature brain to injury, numerous novel animal models have been developed over the past decade. WMD can be induced by antenatal or postnatal administration of microbes (E. coli or Gardnerella vaginalis), virus (border disease virus) or bacterial products (lipopolysaccharide, LPS). Alternatively, various hypoperfusion paradigms or administration of excitatory amino acid receptor agonists (excitotoxicity models) can be used. Irrespective of which insult is utilized, the maturational age of the CNS and choice of species seem critical. Generally, lesions with similarity to human WMD, with respect to distribution and morphological characteristics, are easier to induce in gyrencephalic species (rabbits, dogs, cats and sheep) than in rodents. Recently, however, models have been developed in rats (PND 1,7), using either bilateral carotid occlusion or combined hypoxia-ischemia, that produce predominantly white matter lesions. LPS is the infectious agent most often used to produce WMD in immature dogs, cats, or fetal sheep. The mechanism whereby LPS induces brain injury is not completely understood but involves activation of toll-like receptor 4 on immune cells with initiation of a generalized inflammatory response resulting in systemic hypoglycemia, perturbation of coagulation, cerebral hypoperfusion, and activation of inflammatory cells in the CNS. LPS and umbilical cord occlusion both produce WMD with quite similar distribution in 65% gestational sheep. The morphological appearance is different, however, with a more pronounced infiltration of inflammatory cells into the brain and focal microglia/macrophage ("inflammatory WMD") in response to LPS compared to hypoperfusion evoking a more diffuse microglial response usually devoid of cellular infiltrates ("ischemic WMD"). Furthermore, low doses of LPS that by themselves have no adverse effects in 7-day-old rats (maturation corresponding to the near term human fetus), dramatically increase brain injury to a subsequent hypoxic-ischemic challenge, implicating that bacterial products can sensitize the immature CNS. Contrary to this finding, other bacterial agents like lipoteichoic acid were recently shown to induce tolerance of the immature brain suggesting that the innate immune system may respond differently to various ligands, which needs to be further explored. MRDD Research Reviews 2002;8:30,38. © 2002 Wiley-Liss, Inc. [source]

Effect of Maternal Nutrient Restriction in Early Gestation on Responses of the Hypothalamic-Pituitary-Adrenal Axis to Acute Isocapnic Hypoxaemia in Late Gestation Fetal Sheep

Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep,

HEPATOLOGY, Issue 6 2007
Jason Chamberlain
Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell-based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donor-derived hepatocytes and their pattern of distribution throughout the parenchyma of the recipient's liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n = 6) or intrahepatic (n = 6) route into preimmune fetal sheep. The animals were analyzed 56,70 days after transplantation by immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% ± 3.5% versus 2.6% ± 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. Conclusion: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells. (HEPATOLOGY 2007.) [source]

Fetal and offspring arrhythmia following exposure to nicotine during pregnancy

JOURNAL OF APPLIED TOXICOLOGY, Issue 1 2010
Yu Feng
Abstract Although recent studies have demonstrated prenatal nicotine can increase cardiovascular risk in the offspring, it is unknown whether exposure to nicotine during pregnancy also may be a risk for development of arrhythmia in the offspring. In addition, in previous studies of fetal arrhythmia affected by smoking, only two patterns, bradycardia and tachycardia, were observed. The present study examined acute effects of maternal nicotine on the fetal arrhythmia in utero, and chronic influence on offspring arrhythmia at adult stage following prenatal exposure to nicotine. Nicotine was administered to pregnant ewes and rats. In the fetal sheep, intravenous nicotine not only induced changes of fetal heart rate, but also caused cardiac cycle irregularity, single and multiple dropped cardiac cycles. Although maternal nicotine had no influence on fetal blood pH, lactic acid, hemocrit, Na+, K+ levels and plasma osmolality, fetal blood PO2 levels were significantly decreased following maternal nicotine in ewes. In offspring rats at 4,5 months after birth, prenatal exposure to nicotine significantly increased heart rate and premature ventricular contraction in restraint stress. In addition, arrhythmias induced by injection of nicotine were higher in the offspring prenatal exposure to nicotine in utero. The results provide new evidence that exposure to nicotine in pregnancy can cause fetal arrhythmia in various patterns besides tachycardia and bradycardia, the possible mechanisms for nicotine-induced fetal arrhythmia included in utero hypoxia. Importantly, following exposure to nicotine significantly increased risk of arrhythmia in the adult offspring. The finding offers new insight for development of cardiac rhythm problems in fetal origins. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Appetite Regulatory Neuropeptides are Expressed in the Sheep Hypothalamus Before Birth

JOURNAL OF NEUROENDOCRINOLOGY, Issue 6 2004
B. S. Mühlhäusler
Abstract In the adult, a hypothalamic neural network acts to maintain energy balance in response to nutritional feedback from the periphery. Although there is an immediate requirement for this system to be functional at birth, it is unknown whether the components of this central neural network are expressed in the developing brain before birth. We therefore examined in the fetal sheep hypothalamus during late gestation gene expression for leptin receptor (OB-Rb) and neuropeptides that regulate energy balance in the adult. Brains were collected from fetal sheep at 110 days (n = 12) and 140 days of gestation (n = 5) (term = 150 days) and gene expression was detected in all hypothalami using in situ hybridization with radiolabelled riboprobes for OB-Rb, neuropeptide Y (NPY), agouti-related peptide, pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript (CART). All mRNAs were expressed in the arcuate nucleus of fetuses at both time points. Additional sites of mRNA expression were the dorsomedial hypothalamus (DMH) for NPY, the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and lateral hypothalamic area for CART, and the DMH, PVN and VMH for OB-Rb. We have therefore demonstrated that adult-like localization of gene expression for OB-Rb and key appetite regulatory neuropeptides is established in the ovine hypothalamus before birth. Thus, the fetus possesses a central appetite regulatory neural network with the potential to respond to changes in nutrient supply, which could impact on energy balance regulation both before and after birth. [source]

Divergent effects of ephedrine and phenylephrine on cardiovascular hemodynamics of near-term fetal sheep exposed to hypoxemia and maternal hypotension

ACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 7 2007
T. Erkinaro
Background:, We hypothesized that the administration of ephedrine and phenylephrine for maternal hypotension modifies cardiovascular hemodynamics in near-term sheep fetuses. Methods:, At 115,136 days of gestation, chronically instrumented, anesthetized ewes with either normal placental function or increased placental vascular resistance after placental embolization were randomized to receive boluses of ephedrine (n = 12) or phenylephrine (n = 12) for epidural-induced hypotension after a short period of hypoxemia. Fetal cardiovascular hemodynamics were assessed by Doppler ultrasonography at baseline, during hypotension and after vasopressor treatment. Results:, During hypotension, fetal PO2 decreased and proximal branch pulmonary arterial and pulmonary venous vascular impedances increased. Additionally, in the embolized fetuses, the time-velocity integral ratio between the antegrade and retrograde blood flow components of the aortic isthmus decreased. These parameters were restored to baseline conditions by ephedrine but not by phenylephrine. With phenylephrine, weight-indexed left ventricular cardiac output and ejection force decreased in the non-embolized fetuses, and the proportion of isovolumetric contraction time of the total cardiac cycle was elevated in the embolized fetuses. Conclusions:, After exposure to hypoxemia and maternal hypotension, ephedrine restored all fetal cardiovascular hemodynamic parameters to baseline. Phenylephrine did not reverse fetal pulmonary vasoconstriction or the relative decrease in the net forward flow through the aortic isthmus observed in fetuses with increased placental vascular resistance. Moreover, fetal left ventricular function was impaired during phenylephrine administration. [source]

Surfactant protein expression is increased in the ipsilateral but not contralateral lungs of fetal sheep with left-sided diaphragmatic hernia,

PEDIATRIC PULMONOLOGY, Issue 4 2005
Marcus G. Davey PhD
Abstract Congenital diaphragmatic hernia (CDH) impairs fetal lung growth and increases the density of alveolar epithelial type 2 (AE2) cells. There is controversy whether surfactant protein (SP) expression is altered in CDH. The primary aim of this study was to assess SP expression (mRNA and protein) in the left and right lungs of fetal sheep with and without a diaphragmatic hernia (DH). Left-sided DH was created in four fetal sheep at 65 days of gestational age (g.a.). Sham-operated animals were used as controls. At 138 days g.a., lungs were harvested and the following parameters were measured: SP-A, -B, and -C mRNA expression (Northern blot), SP-A and -B expression (Western blot), and AE2 cell density (immunohistochemistry). The lung weight-to-body weight ratio was reduced by 42% in DH animals. The left-to-right lung weight ratio was lower in DH animals (0.47,±,0.03 vs. 0.69,±,0.03), indicative of asymmetric lung growth. SP-A, -B, and -C mRNA expression were increased by 61.7%, 32.9%, and 75.5%, respectively, in the left lungs of DH animals. SP-A and SP-B were also increased in DH. In the right lung, SP expression (mRNA and protein) was not different between groups. AE2 cell density was higher (by 67%) in the left but not right lungs of DH animals. Although DH in fetal sheep results in significant lung hypoplasia, SP expression is not reduced. On the contrary, SP expression was increased in the ipsilateral lung of fetuses with left-sided DH. Furthermore, AE2 cell density is increased in DH, suggesting that the increase in SP mRNA and protein levels is due to increases AE2 cell number. Our data further support the premise that fetal lung hypoplasia favors an AE2 phenotype. Pediatr Pulmonol. 2005; 39:359,367. © 2005 Wiley-Liss, Inc. [source]

Stimulation of lung growth in fetuses with lung hypoplasia leads to altered postnatal lung structure in sheep

PEDIATRIC PULMONOLOGY, Issue 4 2001
M.G. Davey PhD
Abstract Increased lung expansion in the fetus stimulates lung growth and is being trialed clinically to reverse severe fetal lung hypoplasia. Our aim was to examine the effects of increased fetal lung expansion in the presence of lung hypoplasia on lung structure in sheep at term and 8 weeks after birth. Lung hypoplasia was induced in 15 fetal sheep by continuous drainage of tracheal fluid, commencing at ,113 days of gestation (term, ,148 days). In 10 of these fetuses, tracheal obstruction (TO) was performed from 137,147 days of gestation (treated lung hypoplasia, TLH), while lung liquid drainage continued until term in the remaining 5 fetuses (untreated lung hypoplasia, ULH). Lung tissues were obtained from 5 TLH, 5 ULH, and 5 control lambs at birth, and from 5 TLH and 5 control lambs at 8 weeks after birth. At birth, alveolar number, surface area, and interalveolar wall thickness were not different between TLH and control lambs, whereas airspace diameter was greater in TLH lambs (72.7,±,3.0 ,m) than in controls (58.4,±,4.3 ,m). Diameters of airspaces were not different between ULH and control lambs; however, alveolar numbers and surface area were reduced, while interalveolar wall thickness was increased in ULH lambs compared to controls. At 8 weeks after birth, alveolar number (928.0,±,66.1,×,106) and surface area (30.3,±,2.2 m2) in TLH lambs were lower, whereas interalveolar wall thickness (83.0,±,3.1,m) was greater than in control lambs (2,263.6,±,261.6,×,106, 46.7,±,4.8 m2, and 68.6,±,2.1 ,m, respectively). Our data show that TO restores most aspects of lung structure to normal in fetuses with lung hypoplasia but leads to altered alveolar development. The presence of fewer, larger alveoli in postnatal TLH animals may predispose these animals to respiratory complications during later life. Pediatr Pulmonol. 2001; 32:267,276. © 2001 Wiley-Liss, Inc. [source]

Role of leptin in the regulation of growth and carbohydrate metabolism in the ovine fetus during late gestation

THE JOURNAL OF PHYSIOLOGY, Issue 9 2008
Alison J. Forhead
Leptin is an important regulator of appetite and energy expenditure in adulthood, although its role as a nutritional signal in the control of growth and metabolism before birth is poorly understood. This study investigated the effects of leptin on growth, carbohydrate metabolism and insulin signalling in fetal sheep. Crown,rump length-measuring devices and vascular catheters were implanted in 12 sheep fetuses at 105,110 days of gestation (term 145 ± 2 days). The fetuses were infused i.v. either with saline (0.9% NaCl; n= 6) or recombinant ovine leptin (0.5,1.0 mg kg,1 day,1; n= 6) for 5 days from 125 to 130 days when they were humanely killed and tissues collected. Leptin receptor mRNA and protein were expressed in fetal liver, skeletal muscle and perirenal adipose tissue. Throughout infusion, plasma leptin in the leptin-infused fetuses was 3- to 5-fold higher than in the saline-infused fetuses, although plasma concentrations of insulin, glucose, lactate, cortisol, catecholamines and thyroid hormones did not differ between the groups. Leptin infusion did not affect linear skeletal growth or body, placental and organ weights in utero. Hepatic glycogen content and activities of the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase in the leptin-infused fetuses were lower than in the saline-infused fetuses by 44, 48 and 36%, respectively; however, there were no differences in hepatic glycogen synthase activity or insulin signalling protein levels. Therefore, before birth, leptin may inhibit endogenous glucose production by the fetal liver when adipose energy stores and transplacental nutrient delivery are sufficient for the metabolic needs of the fetus. These actions of leptin in utero may contribute to the development of neonatal hypoglycaemia in macrosomic babies of diabetic mothers. [source]

Hypothalamic input is required for development of normal numbers of thyrotrophs and gonadotrophs, but not other anterior pituitary cells in late gestation sheep

THE JOURNAL OF PHYSIOLOGY, Issue 4 2008
Eva Szarek
To evaluate the hypothalamic contribution to the development of anterior pituitary (AP) cells we surgically disconnected the hypothalamus from the pituitary (hypothalamo-pituitary disconnection, HPD) in fetal sheep and collected pituitaries 31 days later. Pituitaries (n= 6 per group) were obtained from fetal sheep (term = 147 ± 3 days) at 110 days (unoperated group) of gestation and at 141 days from animals that had undergone HPD or sham surgery at 110 days. Cells were identified by labelling pituitary sections with antisera against the six AP hormones. Additionally, we investigated the colocalization of glycoprotein hormones. The proportions of somatotrophs and corticotrophs were unchanged by age or HPD. Lactotrophs increased 80% over time, but the proportion was unaffected by HPD. Thyrotrophs, which were unaffected by age, increased 70% following HPD. Gonadotrophs increased with gestational age (LH+ cells 55%; FSH+ cells 19-fold), but this was severely attenuated by HPD. We investigated the possible existence of a reciprocal effect of HPD on multipotential glycoprotein-expressing cells. Co-expression of LH and TSH was extremely rare (< 1%) and unchanged over the last month of gestation or HPD. The increase of gonadotrophs expressing FSH only or LH and FSH was attenuated by HPD. Therefore, the proportions of somatotrophs, lactotrophs and corticotrophs are regulated independently of hypothalamic input in the late gestation fetal pituitary. In marked contrast, the determination of the thyrotroph and gonadotroph lineages over the same time period is subject to complex mechanisms involving hypothalamic factors, which inhibit differentiation and/or proliferation of thyrotrophs, but stimulate gonadotrophs down the FSH lineage. Development of a distinct population of gonadotrophs, expressing only LH, appears to be subject to alternative mechanisms. [source]

Impact of glucose infusion on the structural and functional characteristics of adipose tissue and on hypothalamic gene expression for appetite regulatory neuropeptides in the sheep fetus during late gestation

THE JOURNAL OF PHYSIOLOGY, Issue 1 2005
B. S. Mühlhäusler
In the present study, our aim was to determine whether intrafetal glucose infusion increases fetal adiposity, synthesis and secretion of leptin and regulates gene expression of the ,appetite regulatory' neuropeptides neuropepetide Y (NPY), agouti-related peptide (AGRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) and receptors (leptin receptor (OB-Rb) and melancortin 3 receptor (MC3R)) within the fetal hypothalamus. Glucose (50% dextrose in saline) or saline was infused (7.5 ml h,1) into fetal sheep between 130 and 140 days gestation (term = 150 ± 3 days gestation). Glucose infusion increased circulating glucose and insulin concentrations, mean lipid locule size (532.8 ± 3.3 ,m2versus 456.7 ± 14.8 ,m2) and total unilocular fat mass (11.7 ± 0.6 g versus 8.9 ± 0.6 g) of the perirenal fat depot. The expression of OB-Rb mRNA was higher in the ventromedial nucleus compared to the arcuate nucleus of the hypothalamus in both glucose and saline infused fetuses (F= 8.04; P < 0.01) and there was a positive correlation between expression of OB-Rb and MC3R mRNA in the arcuate nucleus (r= 0.81; P < 0.005). Glucose infusion increased mRNA expression for POMC, but not for the anorectic neuropeptide CART, or the orexigenic neuropeptides NPY and AGRP, in the arcuate nucleus of the fetal hypothalamus. These findings demonstrate that increased circulating glucose and insulin regulate gene expression of the neuropeptides within the fetal hypothalamus that are part of the neural network regulating energy balance in adult life. [source]

Effects of low dose dexamethasone treatment on basal cardiovascular and endocrine function in fetal sheep during late gestation

THE JOURNAL OF PHYSIOLOGY, Issue 2 2002
Andrew J. W. Fletcher
This study investigated the effects on ovine fetal basal cardiovascular and endocrine functions of fetal intravenous dexamethasone treatment, resulting in circulating concentrations that were one-fifth of the values measured clinically in human infants following maternal antenatal glucocorticoid therapy. Between 117-120 days gestation (dGA; term: ca 145 dGA), 26 Welsh Mountain sheep fetuses were surgically prepared under general anaesthesia with vascular catheters and a Transonic flow probe positioned around a femoral artery. At 125 ± 1 dGA, fetuses were infused with dexamethasone (2.06 ± 0.13 ,g kg,1 h,1i.v.; n= 13) or saline (n= 13) for 48 h. Daily fetal arterial blood samples were taken and cardiovascular data were recorded continuously (data acquisition system). Pressor, vasoconstrictor and chronotropic responses to exogenously administered doses of phenylephrine, angiotensin II and arginine vasopressin (AVP) were determined at 124 ± 1 (pre-infusion), 126 ± 1 (during infusion) and 128 ± 1 (post-infusion) dGA. Fetal cardiac baroreflex curves were constructed using peak pressor and heart rate responses to phenylephrine. Dexamethasone treatment elevated fetal mean arterial blood pressure by 8.1 ± 1.0 mmHg (P < 0.05), increased femoral vascular resistance by 0.65 ± 0.12 mmHg (ml min,1),1 (P < 0.05), depressed plasma noradrenaline concentrations and produced a shift in set-point, but not sensitivity, of the cardiac baroreflex (P < 0.05). Elevations in fetal arterial blood pressure, but not femoral vascular resistance and the shift in baroreflex set-point, persisted at 48 h following dexamethasone treatment. By 48 h following dexamethasone infusion, basal plasma noradrenaline concentration was restored, whilst plasma adrenaline and neuropeptide Y (NPY) concentrations were enhanced, compared with controls (P < 0.05). Fetal dexamethasone treatment did not alter the fetal pressor or femoral vasoconstrictor responses to adrenergic, vasopressinergic or angiotensinergic agonists. These data show that fetal treatment with low concentrations of dexamethasone modifies fetal basal cardiovascular and endocrine functions. Depending on the variable measured, these changes may reverse, persist or become enhanced by 48 h following the cessation of treatment. [source]