The neurosteroid tetrahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety and alters the release and gene expression of corticotropin-releasing hormone in the rat hypothalamus.
Patchev VK, Shoaib M, Holsboer F, Almeida OF.Department of Neuroendocrinology, Max Planck Institute for Psychiatry, Munich, Germany.
The ring-A-reduced progesterone derivative 5 alpha-pregnan-3 alpha-ol-20-one (tetrahydroprogesterone) is synthesized under normal physiological conditions in the brain and is a potent modulator of the GABA receptor.
This neurosteroid has significant sedative and anxiolytic properties.
Corticotropin-releasing hormone plays a major role in stress-induced activation of the hypothalamo-pituitary-adrenal axis, and sustained hyperactivity of hypothalamic corticotropin-releasing hormone-producing neurons may be causally related to both, increased pituitary-adrenal secretion and behavioural symptoms observed in anxiety and affective disorders. We investigated the effect of tetrahydroprogesterone on corticotropin-releasing hormone-induced anxiety, the basal and methoxamine-stimulated release of corticotropin-releasing hormone from hypothalamic organ explants in vitro, and adrenalectomy-induced up-regulation of the gene expression of corticotropin-releasing hormone in the hypothalamic paraventricular nucleus in rats. At doses of 5 and 10 micrograms i.c.v., tetrahydroprogesterone counteracted the anxiogenic action of 0.5 microgram of corticotropin-releasing hormone.
Tetrahydroprogesterone did not alter the basal release of corticotropin-releasing hormone in vitro, but suppressed the stimulatory effect of the alpha 1-adrenergic agonist methoxamine on this parameter. Measurements of the steady-state levels of mRNA coding for corticotropin-releasing hormone by quantitative in situ-hybridization histochemistry revealed that tetrahydroprogesterone was equipotent with corticosterone in preventing adrenalectomy-induced up-regulation of peptide gene expression.
Systemic administration of tetrahydroprogesterone also restrained adrenalectomy-induced thymus enlargement.
These results demonstrate that tetrahydroprogesterone has anxiolytic effects that are mediated through interactions with hypothalamic corticotropin-releasing hormone in both, genomic and non-genomic fashions.
http/www.ncbi.nlm.nih.gov/pubmed/7816204
Allopregnanolone (tetrahydroprogesterone)
From Wikipedia, the free encyclopedia
Allopregnanolone, also known as 3α,5α-tetrahydroprogesterone or THP, is an important neurosteroid in the human brain.
It is a metabolite of progesterone and a barbiturate-like modulator of central gamma-aminobutyric acid (GABA) receptors that modify a range of behaviors, including the stress response.
The 5β epimer of this compound is known as pregnanolone, and has very similar properties to allopregnanolone.
Both compounds are found endogenously and have similar hypnotic and anxiolytic effects.
http/en.wikipedia.org/wiki/Allopregnanolone
Saturday, October 10, 2009
Tetrahydroprogesterone Attenuates the Endocrine Response to Stress
Neuropsychopharmacology (1996) 15 533-540.
The Neurosteroid Tetrahydroprogesterone Attenuates the Endocrine Response to Stress and Exerts Glucocorticoid-like Effects on Vasopressin Gene Transcription in the Rat Hypothalamus
V K Patchev MD, Ph.D, A H S Hassan B.V.Sc, Ph.D, F Holsboer MD, Ph.D and O F X Almeida Ph.D From the Department of Neuroendocrinology, Max Planck Institute of Psychiatry, Clinical Institute, Munich, GermanyCorrespondence: V K Patchev, Department of Neuroendocrinology, Max Planck Institute of Psychiatry, Clinical Institute, Kraepelinstr. 2, 80804 Munich, Germany. E-mail: patchev@mpipsykl.mpg.de
ABSTRACT
The neurosteroid tetrahydroprogesterone (5-pregnan-3-ol-20-one, allopregnanolone, THP), has been previously shown to counteract the anxiogenic effects of corticotropin-releasing hormone (CRH) and to interfere with noradrenergic and corticosteroid-mediated regulation of CRH release and gene transcription.
Those observations indicated that, besides its sedative and analgesic activity, THP may also affect the neuroendocrine response to stress in a mode resembling that of corticosteroids. To examine this possibility, we compared the ability of THP, its precursor progesterone (P4), and the glucocorticoids dexamethasone (DEX) and corticosterone (CORT) to influence the pituitary-adrenal response to acute emotional stress and the adrenalectomy-induced increase in the gene transcription of the stress-related peptide arginine vasopressin (AVP) and of corticosteroid receptors (MR and GR) in the brain. Pretreatment of rats with a single dose of THP or P4 (50 g/kg) significantly attenuated the elevation of plasma adrenocorticotropin (ACTH) and serum corticosterone after emotional stress; both steroids were, however, less potent than a similar dose of DEX. Administration of 1 mg of THP, CORT, or P4 to adrenalectomized (ADX) rats attenuated the increase in AVP mRNA levels in the ventromedial subdivision of the hypothalamic paraventricular nucleus (PVN), as compared with vehicle-treated ADX rats. However, whereas CORT and P4 influenced the ADX-induced increase in the transcription of both types of corticosteroid receptors in the hippocampus, these were unaffected by THP. In contrast to the glucocorticoids, THP and P4 failed to decrease plasma ACTH levels in rats deprived of endogenous steroids.
These results demonstrate that the neurosteroid THP and its precursor P4 resemble glucocorticoids in their suppression of the pituitary-adrenal response to emotional stress;
however, THP influences the transcription of glucocorticoid-responsive genes in brain structures involved in the regulation of the hypothalamo-pituitary-adrenal system in a fashion that is quite distinct from that obtained with glucocorticoids.
Ó American College of NeuropsychopharmacologyKeywords: Neurosteriods; Progesterone; Glucocorticoids; Vasopressin; Corticosteroid receptors; Stress
http://www.nature.com/npp/journal/v15/n6/abs/1380502a.html
The Neurosteroid Tetrahydroprogesterone Attenuates the Endocrine Response to Stress and Exerts Glucocorticoid-like Effects on Vasopressin Gene Transcription in the Rat Hypothalamus
V K Patchev MD, Ph.D, A H S Hassan B.V.Sc, Ph.D, F Holsboer MD, Ph.D and O F X Almeida Ph.D From the Department of Neuroendocrinology, Max Planck Institute of Psychiatry, Clinical Institute, Munich, GermanyCorrespondence: V K Patchev, Department of Neuroendocrinology, Max Planck Institute of Psychiatry, Clinical Institute, Kraepelinstr. 2, 80804 Munich, Germany. E-mail: patchev@mpipsykl.mpg.de
ABSTRACT
The neurosteroid tetrahydroprogesterone (5-pregnan-3-ol-20-one, allopregnanolone, THP), has been previously shown to counteract the anxiogenic effects of corticotropin-releasing hormone (CRH) and to interfere with noradrenergic and corticosteroid-mediated regulation of CRH release and gene transcription.
Those observations indicated that, besides its sedative and analgesic activity, THP may also affect the neuroendocrine response to stress in a mode resembling that of corticosteroids. To examine this possibility, we compared the ability of THP, its precursor progesterone (P4), and the glucocorticoids dexamethasone (DEX) and corticosterone (CORT) to influence the pituitary-adrenal response to acute emotional stress and the adrenalectomy-induced increase in the gene transcription of the stress-related peptide arginine vasopressin (AVP) and of corticosteroid receptors (MR and GR) in the brain. Pretreatment of rats with a single dose of THP or P4 (50 g/kg) significantly attenuated the elevation of plasma adrenocorticotropin (ACTH) and serum corticosterone after emotional stress; both steroids were, however, less potent than a similar dose of DEX. Administration of 1 mg of THP, CORT, or P4 to adrenalectomized (ADX) rats attenuated the increase in AVP mRNA levels in the ventromedial subdivision of the hypothalamic paraventricular nucleus (PVN), as compared with vehicle-treated ADX rats. However, whereas CORT and P4 influenced the ADX-induced increase in the transcription of both types of corticosteroid receptors in the hippocampus, these were unaffected by THP. In contrast to the glucocorticoids, THP and P4 failed to decrease plasma ACTH levels in rats deprived of endogenous steroids.
These results demonstrate that the neurosteroid THP and its precursor P4 resemble glucocorticoids in their suppression of the pituitary-adrenal response to emotional stress;
however, THP influences the transcription of glucocorticoid-responsive genes in brain structures involved in the regulation of the hypothalamo-pituitary-adrenal system in a fashion that is quite distinct from that obtained with glucocorticoids.
Ó American College of NeuropsychopharmacologyKeywords: Neurosteriods; Progesterone; Glucocorticoids; Vasopressin; Corticosteroid receptors; Stress
http://www.nature.com/npp/journal/v15/n6/abs/1380502a.html
Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides
Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides.
Turnbull AV, Rivier C.Clayton Foundation Laboratory of Peptide Biology, Salk Institute, La Jolla, California 92037, USA.
Corticotropin-releasing factor (CRF) is a 41-amino acid neuropeptide, which is recognized as a critical mediator of complimentary, stress-related endocrine, autonomic, and behavioral responses in mammalian species.
CRF belongs to a family of structurally related peptides including frogskin sauvagine and fish urotensin I.
The effects of CRF and related peptides are mediated by two distinct receptors, which differ in their anatomical distribution, as well as in their pharmacological characteristics.
In addition, CRF is bound with high affinity by a CRF binding protein (CRF-BP), which is a putative inhibitor of CRF action.
CRF is probably not the sole endogenous ligand for CRF receptors or the CRF-BP, since a second mammalian member of the CRF family, urocortin, has recently been identified.
This article describes recent findings with respect to CRF, its receptors, binding protein, and CRF-related peptides, which provide further insights into the role and mechanisms of CRF action in stress responses.
PMID: 9142133 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/pubmed/9142133
Turnbull AV, Rivier C.Clayton Foundation Laboratory of Peptide Biology, Salk Institute, La Jolla, California 92037, USA.
Corticotropin-releasing factor (CRF) is a 41-amino acid neuropeptide, which is recognized as a critical mediator of complimentary, stress-related endocrine, autonomic, and behavioral responses in mammalian species.
CRF belongs to a family of structurally related peptides including frogskin sauvagine and fish urotensin I.
The effects of CRF and related peptides are mediated by two distinct receptors, which differ in their anatomical distribution, as well as in their pharmacological characteristics.
In addition, CRF is bound with high affinity by a CRF binding protein (CRF-BP), which is a putative inhibitor of CRF action.
CRF is probably not the sole endogenous ligand for CRF receptors or the CRF-BP, since a second mammalian member of the CRF family, urocortin, has recently been identified.
This article describes recent findings with respect to CRF, its receptors, binding protein, and CRF-related peptides, which provide further insights into the role and mechanisms of CRF action in stress responses.
PMID: 9142133 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/pubmed/9142133
Endogenous substance P inhibits the expression of corticotropin-releasing hormone during a chronic inflammatory stress
Endogenous substance P inhibits the expression of corticotropin-releasing hormone during a chronic inflammatory stress
Copyright © 1995
Published by Elsevier Science Inc.H. S. Chowdreyxa*, P. J. Larsent, M. S. Harbuza, S. L. Lightmana and D. S. Jessopa, a Department of Medicine, University of Bristol, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8Hw, U.K.* School of Chemical and Life Sciences, University of Greenwich, London, U.K.t Department of Medical Anatomy, The Panum Institute, University of, Copenhagen, DenmarkRevised 18 September 1995.
Available online 5 April 2000.
Abstract
We have investigated the effects of a chronic inflammatory stress on substance P (SP) levels in the hypothalami of rats given adjuvant-induced arthritis (AA).
Fourteen days after injection of . substance P concentrations in the paraventricular nucleus (PVN) and median eminence/arcuate nucleus were significantly increased.
In AA rats injected intraperitoneally with the specific neurokinin-1 receptor antagonist RP67580, plasma ACTH and corticosterone concentrations were significantly elevated and corticotropin-releasing hormone (CRH) mRNA in the PVN was increased compared to the AA group which received saline alone. The increases in hypothalamic SP in AA, together with the data demonstrating that HPA axis activity is enhanced in AA following injection of a SP antagonist, are consistent with the hypothesis that SP is acting as an inhibitor of CRH expression in this model of chronic inflammatory stress.Author Keywords: substance P; CRH; ACTH; corticosterone; RP67580
http://www.sciencedirect.com/science...d93c9300c9e 5
Copyright © 1995
Published by Elsevier Science Inc.H. S. Chowdreyxa*, P. J. Larsent, M. S. Harbuza, S. L. Lightmana and D. S. Jessopa, a Department of Medicine, University of Bristol, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8Hw, U.K.* School of Chemical and Life Sciences, University of Greenwich, London, U.K.t Department of Medical Anatomy, The Panum Institute, University of, Copenhagen, DenmarkRevised 18 September 1995.
Available online 5 April 2000.
Abstract
We have investigated the effects of a chronic inflammatory stress on substance P (SP) levels in the hypothalami of rats given adjuvant-induced arthritis (AA).
Fourteen days after injection of . substance P concentrations in the paraventricular nucleus (PVN) and median eminence/arcuate nucleus were significantly increased.
In AA rats injected intraperitoneally with the specific neurokinin-1 receptor antagonist RP67580, plasma ACTH and corticosterone concentrations were significantly elevated and corticotropin-releasing hormone (CRH) mRNA in the PVN was increased compared to the AA group which received saline alone. The increases in hypothalamic SP in AA, together with the data demonstrating that HPA axis activity is enhanced in AA following injection of a SP antagonist, are consistent with the hypothesis that SP is acting as an inhibitor of CRH expression in this model of chronic inflammatory stress.Author Keywords: substance P; CRH; ACTH; corticosterone; RP67580
http://www.sciencedirect.com/science...d93c9300c9e 5
Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird
Am J Physiol Regul Integr Comp Physiol 281: R994-R1003, 2001; 0363-6119/01 $5.00
Vol. 281, Issue 3, R994-R1003, September 2001
Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird
Luke Remage-Healey and L. Michael Romero
Department of Biology, Tufts University, Medford, Massachusetts 02155
Captive European starlings (Sturnus vulgaris) were exposed to the stress of handling and restraint while corticosterone, glucose, and triglyceride concentrations were monitored in blood plasma.
In saline-injected controls, basal samples were taken within 3 min of disturbance with subsequent samples taken at 40, 70, and 150 min.
This was repeated at two times during the daily cycle (day and night) on two different photoperiods: short and long days.
During both photoperiods, corticosterone concentrations approximately tripled (compared with a sixfold increase in free-living starlings) and triglyceride concentrations decreased 25-45% in response to stress at both times of the day, whereas an ~25% stress-induced hyperglycemia occurred only at night.
Exogenous corticosterone (200 µg), 1.0 or 4.0 IU/kg of insulin, or a combination of corticosterone with each insulin dose was then separately administered to alter the above responses.
Insulin did not affect corticosterone or triglyceride concentrations but resulted in a dose-dependent hypoglycemia of 10-40%. Injected corticosterone resulted in supraphysiological corticosterone concentrations (three- to fivefold higher than normal), yet it did not affect the already altered plasma glucose or triglyceride concentrations.
This suggests that glucose output and triglyceride decreases were already maximal in response to handling and restraint.
However, the low glucose concentrations resulting from exogenous insulin returned to basal quicker with exogenous corticosterone but only during the day. No response to either hormone showed photoperiodic differences.
These data suggest that corticosterone's role in metabolism changes to meet varying energetic demands throughout the day.
daily rhythms; seasonal rhythms; hypoglycemia; energy metabolism
http://ajpregu.physiology.org/cgi/content/abstract/281/3/R994
Vol. 281, Issue 3, R994-R1003, September 2001
Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird
Luke Remage-Healey and L. Michael Romero
Department of Biology, Tufts University, Medford, Massachusetts 02155
Captive European starlings (Sturnus vulgaris) were exposed to the stress of handling and restraint while corticosterone, glucose, and triglyceride concentrations were monitored in blood plasma.
In saline-injected controls, basal samples were taken within 3 min of disturbance with subsequent samples taken at 40, 70, and 150 min.
This was repeated at two times during the daily cycle (day and night) on two different photoperiods: short and long days.
During both photoperiods, corticosterone concentrations approximately tripled (compared with a sixfold increase in free-living starlings) and triglyceride concentrations decreased 25-45% in response to stress at both times of the day, whereas an ~25% stress-induced hyperglycemia occurred only at night.
Exogenous corticosterone (200 µg), 1.0 or 4.0 IU/kg of insulin, or a combination of corticosterone with each insulin dose was then separately administered to alter the above responses.
Insulin did not affect corticosterone or triglyceride concentrations but resulted in a dose-dependent hypoglycemia of 10-40%. Injected corticosterone resulted in supraphysiological corticosterone concentrations (three- to fivefold higher than normal), yet it did not affect the already altered plasma glucose or triglyceride concentrations.
This suggests that glucose output and triglyceride decreases were already maximal in response to handling and restraint.
However, the low glucose concentrations resulting from exogenous insulin returned to basal quicker with exogenous corticosterone but only during the day. No response to either hormone showed photoperiodic differences.
These data suggest that corticosterone's role in metabolism changes to meet varying energetic demands throughout the day.
daily rhythms; seasonal rhythms; hypoglycemia; energy metabolism
http://ajpregu.physiology.org/cgi/content/abstract/281/3/R994
CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production
Regulation of progesterone production in human term trophoblasts in vitro by CRH, ACTH and cortisol (prednisolone)
Udo Jeschke1 , Ioannis Mylonas1, Dagmar-Ulrike Richter2, Ingo Höcker2, Volker Briese2, Antonis Makrigiannakis3 and Klaus Friese1(1) 1st Department of Obstetrics and Gynaecology, Ludwig-Maximilians-University of Munich, Maistrasse 11, 80337 Munich, Germany (2) Department of Obstetrics and Gynecology, University of Rostock, Rostock, Germany (3) Department of Obstetrics & Gynocology, Medical School, University of Crete, Heraklion, 71110, Greece
Received: 13 December 2004
Accepted: 11 January 2005
Published online: 16 April 2005
Abstract Background:
In most mammals, onset of labor is accompanied with progesterone withdrawal.
In humans, cortisol blockade of progesterone is a possible mechanism involved in the initiation of labor.
Therefore, aim of the study was to clarify the effect of CRH, ACTH and cortisol (prednisolone) on the release of progesterone by term trophoblast cells in vitro.
Methods: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a percoll gradient centrifugation step.
Trophoblasts were incubated with CRH, ACTH as well as with prednisolone
Results:
The release of progesterone is decreased in CRH- and ACTH-treated trophoblast cell cultures compared to untreated trophoblast cells.
Addition of prednisolone in varying concentrations leads to an increase of trophoblast progesterone production.
Conclusions:
The results suggest that CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production.
Prednisolone on the other hand showed a stimulating effect on progesterone production in term trophoblast cells in vitro.
Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that CRH and ACTH are directly involved in the auto- or paracrine regulation of this procedure. Keywords CRH - ACTH - Prednisolone - Progesterone production - Trophoblast cells
http/www.springerlink.com/content/hg9txt426r9q2821/
A short luteal phase (is the period that starts at ovulation and ends on the day before the next period) that lasts less than 10 days also indicates low levels of progesterone.
http/www.buzzle.com/articles/low-progesterone.html
Udo Jeschke1 , Ioannis Mylonas1, Dagmar-Ulrike Richter2, Ingo Höcker2, Volker Briese2, Antonis Makrigiannakis3 and Klaus Friese1(1) 1st Department of Obstetrics and Gynaecology, Ludwig-Maximilians-University of Munich, Maistrasse 11, 80337 Munich, Germany (2) Department of Obstetrics and Gynecology, University of Rostock, Rostock, Germany (3) Department of Obstetrics & Gynocology, Medical School, University of Crete, Heraklion, 71110, Greece
Received: 13 December 2004
Accepted: 11 January 2005
Published online: 16 April 2005
Abstract Background:
In most mammals, onset of labor is accompanied with progesterone withdrawal.
In humans, cortisol blockade of progesterone is a possible mechanism involved in the initiation of labor.
Therefore, aim of the study was to clarify the effect of CRH, ACTH and cortisol (prednisolone) on the release of progesterone by term trophoblast cells in vitro.
Methods: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a percoll gradient centrifugation step.
Trophoblasts were incubated with CRH, ACTH as well as with prednisolone
Results:
The release of progesterone is decreased in CRH- and ACTH-treated trophoblast cell cultures compared to untreated trophoblast cells.
Addition of prednisolone in varying concentrations leads to an increase of trophoblast progesterone production.
Conclusions:
The results suggest that CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production.
Prednisolone on the other hand showed a stimulating effect on progesterone production in term trophoblast cells in vitro.
Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that CRH and ACTH are directly involved in the auto- or paracrine regulation of this procedure. Keywords CRH - ACTH - Prednisolone - Progesterone production - Trophoblast cells
http/www.springerlink.com/content/hg9txt426r9q2821/
A short luteal phase (is the period that starts at ovulation and ends on the day before the next period) that lasts less than 10 days also indicates low levels of progesterone.
http/www.buzzle.com/articles/low-progesterone.html
Effects of Psychological Stress and Alprazolam on Development of Oral Candidiasis in Rats
Note: ALPRAZOLAM = XANAX
Clin Diagn Lab Immunol. 2002 July; 9(4): 852–857.
doi: 10.1128/CDLI.9.4.852-857.2002.
PMCID: PMC120028
Copyright © 2002, American Society for Microbiology
M. J. Núñez, J. Balboa, P. Riveiro, D. Liñares, P. Mañá, M. Rey-Méndez, A. Rodríguez-Cobos, J. A. Suárez-Quintanilla, L. A. García-Vallejo, and M. Freire-Garabal*
Neuroimmunology Laboratory, Department of Pharmacology, School of Medicine, University of Santiago de Compostela, 15705-Santiago de Compostela, Spain
*Corresponding author. Mailing address: Neuroimmunology Laboratory, Department of Pharmacology, School of Medicine, University of Santiago de Compostela, 15705-Santiago de Compostela, Spain. Phone: 34 600 942 256. Fax: 34 981 573 191. E-mail: fffregar@usc.es
.
Received January 29, 2001; Revised May 16, 2001; Accepted April 23, 2002.
Effects of Psychological Stress and Alprazolam on Development of Oral Candidiasis in Rats
Abstract
Psychological stress has been found to suppress cell-mediated immune responses that are important in limiting the proliferation of Candida albicans. Since anxiolytic drugs can restore cellular immunity in rodents exposed to stress conditions, we designed experiments conducted to evaluate the effects of alprazolam (1 mg/kg of body weight/day), a central benzodiazepine anxiolytic agonist, on the development of oral candidiasis in Sprague-Dawley rats exposed to a chronic auditory stressor. Animals were submitted to surgical hyposalivation in order to facilitate the establishment and persistence of C. albicans infection. Application of stress and treatment with drugs (placebo or alprazolam) were initiated 7 days before C. albicans inoculation and lasted until the end of the experiments (day 15 postinoculation). Establishment of C. albicans infection was evaluated by swabbing the inoculated oral cavity with a sterile cotton applicator on days 2 and 15 after inoculation, followed by plating on YEPD (yeast extract-peptone-dextrose) agar. Tissue injury was determined by the quantification of the number and type (normal or abnormal) of papillae on the dorsal tongue per microscopic field. A semiquantitative scale was devised to assess the degree of colonization of the epithelium by fungal hyphae. Our results show that stress exacerbates C. albicans infection of the tongues of rats. Significant increases in Candida counts, the percentage of the tongue's surface covered with clinical lesions, the percentage of abnormal papillae, and the colonization of the epithelium by fungal hyphae were found in stressed rats compared to those found in the unstressed rats. Treatment with alprazolam significantly reversed these adverse effects of stress, showing that, besides the psychopharmacological properties of this anxiolytic drug against stress, it has consequences for Candida infection.
Candida albicans is an example of an opportunistic pathogen frequently isolated from the human mouth, yet few carriers develop clinical signs of candidiasis. The most common predisposing factors to oral candidiasis are immunosuppresive therapy, immunoincompetence, and immunodeficiencies, indicating that the host immune system provides a protective mechanism against superficial invasion by Candida.
Several lines of evidence indicate that cell-mediated immunity is important in limiting the proliferation of Candida; thus, this opportunistic human pathogen preferentially causes invasive and disseminated infections in patients with defective phagocytic defenses and serious mucocutaneous infection in patients with deficiencies in T-cell function. Phagocytes appear to protect the host from fungal colonization even in the absence of adaptive immune mechanisms, while as-yet-undefined T-cell-dependent factors seem necessary for the control of C. albicans on body surfaces (31).
In our previous research, we had observed adverse effects of stress on natural and specific immune responses that may predispose the host to more severe Candida infections (22). On the other hand, treatment with benzodiazepines (BZDs), such as alprazolam, was found to attenuate some of the effects of stress on the immune systems of rodents, such as T-cell depletion, the inhibition of the blastogenic and cytotoxic activities of spleen cells (14, 15, 18), impaired delayed type hypersensitivity (38), and defects in phagocytosis (21). We have already tested this drug in laboratory animal models of infection showing a correlation between the immunoprotective effect of alprazolam and the host resistance against bacteria (16) and viruses (19, 20). Despite other known or unknown mechanisms, central pharmacological effects regulating the release of neuroendocrine hormones, such us adrenalcorticotropic hormone (ACTH), should be involved, at least in part, in the effects of alprazolam on immunocompetence. Nevertheless, there is little data on the effects of this compound on the development of fungal infection. In order to further elucidate this relationship, we studied the effects of alprazolam on the development of oral candidiasis in rats exposed to a repeated auditory stressor.
MATERIALS AND METHODS
Animals.
Two-month-old male pathogen-free rats of the Sprague-Dawley strain (Interfauna Iberica, S.A., Barcelona, Spain) weighing 180 to 200 g were used. They were housed individually in filter-top cages and screened for the presence of C. albicans by plating oral swabs on YEPD (yeast extract-peptone-dextrose) agar (Sigma Chemical Co., St. Louis, Mo.) (17, 31). The cages were kept in a temperature-controlled (22 to 24°C) and humidity-controlled animal room, with an alternating light-dark cycle (lights on at 0600 and lights off at 1800) and with food (diet A.03; Panlab, Barcelona, Spain) and sterile water ad libitum.
Procedure.
Following verification that the rats were free of C. albicans, they were randomly divided into six experimental groups of four animals each according to the treatment they were to be submitted to: group 1, control (i.e., no stress or placebo); group 2, unstressed rats injected with placebo; group 3, unstressed rats injected with alprazolam; group 4, stressed rats with no treatment; group 5, stressed rats injected with placebo; group 6, stressed rats injected with alprazolam.
Stress procedure.
The rats were subjected to a broadband noise at 100 db daily for 5 s every minute during either a 1- or 3-h period (at random) around midnight, at the height of the diurnal activity cycle (32). All stressed rats were subjected to the same stress schedule. Unstimulated rats were exposed only to the normal activity of the animal room. Stress application started 7 days before C. albicans inoculation and lasted until the end of experiments (day 15 postinoculation).
Treatment with drugs.
Alprazolam (Upjohn, Kalamazoo, Inc.) was intraperitoneally injected at a dose of 1 mg/kg of body weight in a volume of 1 ml of 1% water solution of carboxymethylcellulose per kg of body weight as a vehicle. Mice in the placebo group were intraperitoneally injected with 1 ml of diluent per kg of body weight. Drugs were administered daily at 9:30 a.m. during all periods of stress application.
Surgical hyposalivation.
As in humans, xerostomia in rats facilitates the establishment and persistence of C. albicans infection in the mouth; therefore, it constitutes a suitable animal model for the study of oral candidiasis (25). Sialoadenectomy in rats causes intense xerostomia, but the minor salivary glands, the main producers of mucin, an important barrier for mucosal permeability and a major source of immunoglobulin A, were preserved. In our experiment, xerostomia was surgically provoked in all rats 1 month before treatment with drugs and stress application were initiated. The rats were anesthetized with 44 mg of ketamine (Ketolar; Parke-Davis, Barcelona, Spain) per kg of body weight and 1 mg of diazepam (Valium; Roche, Madrid, Spain) per kg of body weight (40). The parotid salivary ducts of the animals were ligated, and the submandibular and sublingual salivary glands were surgically removed according to procedures previously described (7, 30, 31).
Source and culture of C. albicans.
The C. albicans organisms used to inoculate the rats were obtained from a patient with erythematous oral candidiasis (17). The Candida strains were grown on YEPD agar plates at room temperature (35). The isolated organisms were identified as C. albicans by a germ tube test and chlamydospore production as described by Schaar et al. (36).
Inoculation of C. albicans.
The C. albicans organisms isolated were prepared for inoculation by suspending colonies in sterile buffered saline and were washed twice by centrifugation before being resuspended in normal saline. The concentration of organisms was adjusted to 3 × 108/ml by optical density at 300 nm (3). The tongues of the animals were swabbed on 2 successive days with a cotton-tipped applicator saturated with 0.1 ml of fresh inoculum (31).
Quantification of C. albicans cells.
Establishment of C. albicans infection was evaluated by swabbing the inoculated oral cavity with a sterile cotton applicator, followed by plating on YEPD agar (25, 31). Samples were collected 2 days after inoculation and at the end of experiment. The cotton applicator was immediately immersed in 0.99 ml of sterile isotonic saline to obtain a dilution of 10−2, and it was agitated for 2 min. This dilution was considered to be 10−2. Dilutions up to 10−5 (0.1 ml) were cultured in duplicate in Sabouraud's dextrose agar at 37°C for 48 h. Candida colonies were counted in plates exhibiting between 30 and 300 colonies. Plates with less than 30 colonies in the 10−2 dilution were considered to have 101 cells (25).
Clinical lesions.
At the end of the experimental period, all animals were sacrificed by asphyxiation in a CO2 atmosphere and were then decapitated. The dorsal tongue was photographed in situ at a magnification of ×10 (3). Clinical lesions were measured with a digital imaging system (Técnicas Médicas MAB, Barcelona, Spain) and expressed as the percentage of the surface area of the tongue (percent area) that was covered with the lesions.
Tissue handling.
The tongues from the rats were hemidissected in the sagittal plane, with half of the lesion immersed in 10% buffered formalin for routine processing and the other half placed in 2.5% glutaraldehyde with 0.1 M Sorensen's phosphate buffer at 4°C (3).
Light microscopy.
Both hematoxylin and eosin and periodic acid-Schiff stains were used. C. albicans infection was assessed with a digital imaging system according to evidence of lesions and hyphal colonization on the dorsal tongue (3, 33). Tissue injury was determined by quantification of the number and type (normal, atrophic, and hypertropic) of papillae per microscopic field (magnification, ×46). A semiquantitative scale was devised to assess the degree of colonization of the epithelium by fungal hyphae. In this scale, the absence of colonization was given a score of 0, while maximal colonization, in excess of 50 hyphae, could be seen in each high-power field (magnification, ×400) was assigned a score of 4. The scores given were 1 for 1 to 5 hyphae, 2 for 6 to 15 hyphae, and 3 for 16 to 50 hyphae. The specimens were examined by one of us, who was blind as to the source. Three high-power fields per sample were examined for the light microscopy experiments.
Scanning electron microscopy preparation.
Following fixation for 24 h, the tissue was rinsed three times in buffer and postfixed in 1% phosphate-buffered osmium tetroxide (pH 7.4) for 1 h. After two buffer rinses, the specimens were dehydrated in ascending concentrations of ethanol, followed by critical point dehydration in a Denton DCP-1 critical point drying apparatus with liquid CO2. The tissue samples were affixed on aluminum stubs with silver conductive paint and were sputter-coated with gold-palladium by using a Hummer VI sputter-coating apparatus (Anatech Electronics, Garfield, N.J.). Specimens were viewed with a Zeiss 910 electron microscope (Zeiss, Oberkochen, Germany) operated at 20 kV (2).
Statistical analysis.
Statistical analysis of the quantitation of C. albicans cells in oral tissue was performed by one-way analysis of variance, followed by Bonferroni's t test. The percentage of areas of clinical lesions was analyzed by Student's t test (25). The Wilcoxon signed-rank sum test for paired comparisons and the Kruskal-Wallis test for multiple comparisons were used to determine the degree of colonization of the epithelium by fungal hyphae (33). Differences were considered significant at P < 0.05.
RESULTS
C. albicans counts at 2 and 15 days after inoculation (Table 1), as well as the percent area of clinical lesions in the dorsal tongue (Table 2), were increased in stressed rats compared with those in unstressed animals (differences, P < class="fig-table-link fig figpopup" style="TEXT-DECORATION: none" onclick="startTarget(this, 'figure', 1024, 800)" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=120028&rendertype=figure&id=f1" hoverintent_t="undefined" jquery1255197605890="2">(Fig.1)1) were observed in stressed animals (differences, P < class="fig-table-link fig figpopup" style="TEXT-DECORATION: none" onclick="startTarget(this, 'figure', 1024, 800)" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=120028&rendertype=figure&id=f2" jquery1255197605890="3">(Fig.2)2) scored higher than untreated controls (differences, P <> 0.05), with the only exception that placebo increased the degree of colonization of the epithelium in unstressed animals. In contrast, treatment with alprazolam significantly (P < 0.05) reversed the adverse effects of stress in all parameters assayed.
Clinically evident lesions and inflammatory changes of the underlying connective tissue were observed 15 days after C. albicans inoculation. The latter were found in all experimental groups, but they were more evident in stressed rats. Animals showed macroscopic focal patchy atrophy of the dorsal tongue papillae. Light microscopy showed localized dense zones of hyphal penetration of the keratin layer in the giant conical papillae and filiform papillae of the dorsal tongue. Microabscesses in the keratin and the superficial spinous layers were observed in association with hyphal invasion. The underlying connective tissue showed a mild chronic inflammatory cell infiltrate. Those papillae that supported the Candida growth appeared shorter and blunter than the surrounding uninfected papillae.
Scanning electron microscopy of the dorsal tongues showed a higher loss of papillae in the giant conical and filiform areas of the specimens together with an increase in the size of the flat central portion of the lesion in stressed rats in comparison with unstressed animals. This adverse effect of stress was also reduced by the administration of alprazolam.
DISCUSSION
Our results show that stress exacerbates C. albicans infection of the tongues of rats. Significant increases in Candida counts, the percent area of clinical lesions, the percent abnormal papillae, and the colonization of the epithelium by fungal hyphae were found in stressed rats compared with those found in unstressed animals. Treatment with alprazolam partially reversed those adverse effects of stress on the development of oral candidiasis. Alprazolam was found to reverse many of the effects of stress on C. albicans infection of the tongues of rats, including Candida counts, the percent area of clinical lesions, the percent abnormal papillae, and the colonization of the epithelium by fungal hyphae.
Clinical and experimental observations indicate that the opportunistic proclivities of this fungus vary considerably, depending on the nature of the immunological defect of the victim.
Patients with qualitative or quantitative defects of phagocytes are mainly prone to the invasive form of this mycosis (10, 11, 37).
In contrast, defective T-cell-mediated immunity has been specifically associated with thrush and other forms of candidiasis limited to mucocutaneous surfaces (11, 12, 24, 26). Krause and Schaffner (28) demonstrated that cyclosporine, a relatively selective suppressor of T-cell-mediated immunity and NK cell activity, promoted the formation of thrushlike lesions on cyst surfaces and impeded the elimination of C. albicans from such lesions, but it had no effect on systemic candidiasis induced by intravenous inoculation.
Our results are in line with the previous literature on the stress-induced modulation of the immune system. Changes in murine splenic cytotoxic activities, mediated by NK cells and cytotoxic T lymphocytes have been reported (10-12, 24, 26, 32, 37). Stress also interferes with the activity of phagocytosis and T-cell-dependent antibody responses (21, 28).
The mechanism by which stress inhibits the cellular immune response has been widely studied. A molecular basis for bidirectional communication between the immune and neuroendocrine systems has been described previously (5). Cell-to-cell communication between the immune and the neuroendocrine systems is primarily mediated by hormones and neuropeptides that reach lymphoid organs and cells through the vascular system or directly through the autonomic connections between nerve endings and lymphoid organs (1, 8). Receptor sites are present in lymphoid cells for many hormones and neurotransmitters (6, 39). A number of molecules produced by cells of the nervous system such as ACTH, PRL, opioid peptides, GH, TSH, dynorphin, dopamine, and others have been shown to have the ability to modulate immune functions.
On the other hand, humoral factors generated by the immune system, such as thymic peptides and lymphokines, modulate neuroendocrine functions. In addition, in the course of lymphocyte activation, lymphoid cells may produce hormonal substances identical to those produced by the hypophysis, such as ACTH, TSH, GH, PRL, gonadotrophin, and β-endorphin (6).
At least one of the neuroendocrine responses to stress, such as the rise in plasma corticosterone concentrations via ACTH secretion, has an easily demonstrable destructive effect on specific cells and tissues that are required for optimal immune defense (4, 34). In our previous studies, we observed a stress-induced increase in ACTH levels proportional to the decrease in T-cell populations (15). Nevertheless, in these studies, we observed that adrenalectomized mice showed a lower pattern of immunosuppression in comparison with sham-operated mice. So, this led us to believe that other neuropeptides and neurotransmitters could be involved in the immunosuppressive response to stress.
The effects of alprazolam, an anxiolytic drug with high affinity for central BZD receptors on the pathogenicity of this opportunistic fungus could be attributed, at least in part, to its well-known protective effects against the immunosuppressive response to the type of stress assayed here. The recovery of the immune state of the victim could decrease the pathogenicity of this opportunistic fungus. In this regard, in our previous studies, we demonstrated that alprazolam reversed the suppressive effects of stress on the activity of phagocytosis, T-cell populations, the blastogenic response of spleen cells, murine splenic cytotoxic activities, mediated by NK cells and CTL. Fride et al. (23) found that low doses (0.02 to 1.0 mg/kg) of alprazolam significantly increased the NK cell activity, mixed leukocyte reactivity, and mitogen-induced lymphocyte proliferation in unstressed mice.
The mechanism of action of BZDs on the immune system remains to be defined. A dual approach has been described at the present time. First, central pharmacological effects related to the central type BZD receptors that facilitate inhibitory GABA neurotransmission in the central nervous system may regulate the release of neuroendocrine hormones involved in the immune response to stress. The ability of alprazolam to decrease the stress-induced increase of ACTH levels (29) has been demonstrated to play a important role in the immunoprotective effects of this drug. Nevertheless, significant immunoenhancing effects of alprazolam were also appreciated in stressed adrenalectomized rats (15), suggesting that the modulatory effect of this BZD agonist on other neurohormones like opioid peptides, PRL, melatonin, TSH, or GH could also be involved (13).
A second aspect of the effects of BZDs is the existence of a BZD receptor with high affinity on immune cells that express the so-called peripheral specificity for BZDs (41). Nevertheless, alprazolam is described in the literature as strict central type ligand of the BZD receptor (29). Alprazolam has potent PAF antagonist properties (27) that seem to affect T-cell, B-cell, and macrophage responses under in vitro conditions (9).
One could ask whether secondary (nonimmune or biochemical) effects of the drug treatment might account for the final observations and whether or not stress might break down the state of tolerance normally associated with Candida infection (as opposed to acting solely as an immunopotentiator). Although these considerations should be taken into account, our previous data concerning the immunomodulatory effects of alprazolam under stress conditions (14, 15, 18, 21, 38) lead us to consider immune changes as the main factor involved on the effects of stress and alprazolam on the evolution of oral candidiasis in rats.
A second question concerns the biological significance of our results. Although our data at present show stress may leave the subject vulnerable to the action of C. albicans and provide evidence of a protective effect of alprazolam on the development of oral candidiasis in rats, the biological significance and health relatedness of these findings should be assessed. In this respect, differences between untreated stressed rats and placebo- or alprazolam-stressed rats are statistically significant, but in some parameters, they are not striking. Moreover, there is a relationship between differences obtained in different determinations, but there is not a mathematical correlation as expected.
The large number of interactions at molecular, cellular, and functional levels between the nervous system and the immune system characterizing the operational compositions and expressions of the neuroimmune network make complex isolation of the pathways in which stress and alprazolam may be involved in the regulation of the host defense mechanisms against infection. Nevertheless, the literature has provided evidence that stress-induced immunosuppression and alprazolam-induced immunoprotection are in a relationship with susceptibility to bacteria (16), virus (20), and, as a conclusion of the present investigation, Candida infection.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=120028
Clin Diagn Lab Immunol. 2002 July; 9(4): 852–857.
doi: 10.1128/CDLI.9.4.852-857.2002.
PMCID: PMC120028
Copyright © 2002, American Society for Microbiology
M. J. Núñez, J. Balboa, P. Riveiro, D. Liñares, P. Mañá, M. Rey-Méndez, A. Rodríguez-Cobos, J. A. Suárez-Quintanilla, L. A. García-Vallejo, and M. Freire-Garabal*
Neuroimmunology Laboratory, Department of Pharmacology, School of Medicine, University of Santiago de Compostela, 15705-Santiago de Compostela, Spain
*Corresponding author. Mailing address: Neuroimmunology Laboratory, Department of Pharmacology, School of Medicine, University of Santiago de Compostela, 15705-Santiago de Compostela, Spain. Phone: 34 600 942 256. Fax: 34 981 573 191. E-mail: fffregar@usc.es
.
Received January 29, 2001; Revised May 16, 2001; Accepted April 23, 2002.
Effects of Psychological Stress and Alprazolam on Development of Oral Candidiasis in Rats
Abstract
Psychological stress has been found to suppress cell-mediated immune responses that are important in limiting the proliferation of Candida albicans. Since anxiolytic drugs can restore cellular immunity in rodents exposed to stress conditions, we designed experiments conducted to evaluate the effects of alprazolam (1 mg/kg of body weight/day), a central benzodiazepine anxiolytic agonist, on the development of oral candidiasis in Sprague-Dawley rats exposed to a chronic auditory stressor. Animals were submitted to surgical hyposalivation in order to facilitate the establishment and persistence of C. albicans infection. Application of stress and treatment with drugs (placebo or alprazolam) were initiated 7 days before C. albicans inoculation and lasted until the end of the experiments (day 15 postinoculation). Establishment of C. albicans infection was evaluated by swabbing the inoculated oral cavity with a sterile cotton applicator on days 2 and 15 after inoculation, followed by plating on YEPD (yeast extract-peptone-dextrose) agar. Tissue injury was determined by the quantification of the number and type (normal or abnormal) of papillae on the dorsal tongue per microscopic field. A semiquantitative scale was devised to assess the degree of colonization of the epithelium by fungal hyphae. Our results show that stress exacerbates C. albicans infection of the tongues of rats. Significant increases in Candida counts, the percentage of the tongue's surface covered with clinical lesions, the percentage of abnormal papillae, and the colonization of the epithelium by fungal hyphae were found in stressed rats compared to those found in the unstressed rats. Treatment with alprazolam significantly reversed these adverse effects of stress, showing that, besides the psychopharmacological properties of this anxiolytic drug against stress, it has consequences for Candida infection.
Candida albicans is an example of an opportunistic pathogen frequently isolated from the human mouth, yet few carriers develop clinical signs of candidiasis. The most common predisposing factors to oral candidiasis are immunosuppresive therapy, immunoincompetence, and immunodeficiencies, indicating that the host immune system provides a protective mechanism against superficial invasion by Candida.
Several lines of evidence indicate that cell-mediated immunity is important in limiting the proliferation of Candida; thus, this opportunistic human pathogen preferentially causes invasive and disseminated infections in patients with defective phagocytic defenses and serious mucocutaneous infection in patients with deficiencies in T-cell function. Phagocytes appear to protect the host from fungal colonization even in the absence of adaptive immune mechanisms, while as-yet-undefined T-cell-dependent factors seem necessary for the control of C. albicans on body surfaces (31).
In our previous research, we had observed adverse effects of stress on natural and specific immune responses that may predispose the host to more severe Candida infections (22). On the other hand, treatment with benzodiazepines (BZDs), such as alprazolam, was found to attenuate some of the effects of stress on the immune systems of rodents, such as T-cell depletion, the inhibition of the blastogenic and cytotoxic activities of spleen cells (14, 15, 18), impaired delayed type hypersensitivity (38), and defects in phagocytosis (21). We have already tested this drug in laboratory animal models of infection showing a correlation between the immunoprotective effect of alprazolam and the host resistance against bacteria (16) and viruses (19, 20). Despite other known or unknown mechanisms, central pharmacological effects regulating the release of neuroendocrine hormones, such us adrenalcorticotropic hormone (ACTH), should be involved, at least in part, in the effects of alprazolam on immunocompetence. Nevertheless, there is little data on the effects of this compound on the development of fungal infection. In order to further elucidate this relationship, we studied the effects of alprazolam on the development of oral candidiasis in rats exposed to a repeated auditory stressor.
MATERIALS AND METHODS
Animals.
Two-month-old male pathogen-free rats of the Sprague-Dawley strain (Interfauna Iberica, S.A., Barcelona, Spain) weighing 180 to 200 g were used. They were housed individually in filter-top cages and screened for the presence of C. albicans by plating oral swabs on YEPD (yeast extract-peptone-dextrose) agar (Sigma Chemical Co., St. Louis, Mo.) (17, 31). The cages were kept in a temperature-controlled (22 to 24°C) and humidity-controlled animal room, with an alternating light-dark cycle (lights on at 0600 and lights off at 1800) and with food (diet A.03; Panlab, Barcelona, Spain) and sterile water ad libitum.
Procedure.
Following verification that the rats were free of C. albicans, they were randomly divided into six experimental groups of four animals each according to the treatment they were to be submitted to: group 1, control (i.e., no stress or placebo); group 2, unstressed rats injected with placebo; group 3, unstressed rats injected with alprazolam; group 4, stressed rats with no treatment; group 5, stressed rats injected with placebo; group 6, stressed rats injected with alprazolam.
Stress procedure.
The rats were subjected to a broadband noise at 100 db daily for 5 s every minute during either a 1- or 3-h period (at random) around midnight, at the height of the diurnal activity cycle (32). All stressed rats were subjected to the same stress schedule. Unstimulated rats were exposed only to the normal activity of the animal room. Stress application started 7 days before C. albicans inoculation and lasted until the end of experiments (day 15 postinoculation).
Treatment with drugs.
Alprazolam (Upjohn, Kalamazoo, Inc.) was intraperitoneally injected at a dose of 1 mg/kg of body weight in a volume of 1 ml of 1% water solution of carboxymethylcellulose per kg of body weight as a vehicle. Mice in the placebo group were intraperitoneally injected with 1 ml of diluent per kg of body weight. Drugs were administered daily at 9:30 a.m. during all periods of stress application.
Surgical hyposalivation.
As in humans, xerostomia in rats facilitates the establishment and persistence of C. albicans infection in the mouth; therefore, it constitutes a suitable animal model for the study of oral candidiasis (25). Sialoadenectomy in rats causes intense xerostomia, but the minor salivary glands, the main producers of mucin, an important barrier for mucosal permeability and a major source of immunoglobulin A, were preserved. In our experiment, xerostomia was surgically provoked in all rats 1 month before treatment with drugs and stress application were initiated. The rats were anesthetized with 44 mg of ketamine (Ketolar; Parke-Davis, Barcelona, Spain) per kg of body weight and 1 mg of diazepam (Valium; Roche, Madrid, Spain) per kg of body weight (40). The parotid salivary ducts of the animals were ligated, and the submandibular and sublingual salivary glands were surgically removed according to procedures previously described (7, 30, 31).
Source and culture of C. albicans.
The C. albicans organisms used to inoculate the rats were obtained from a patient with erythematous oral candidiasis (17). The Candida strains were grown on YEPD agar plates at room temperature (35). The isolated organisms were identified as C. albicans by a germ tube test and chlamydospore production as described by Schaar et al. (36).
Inoculation of C. albicans.
The C. albicans organisms isolated were prepared for inoculation by suspending colonies in sterile buffered saline and were washed twice by centrifugation before being resuspended in normal saline. The concentration of organisms was adjusted to 3 × 108/ml by optical density at 300 nm (3). The tongues of the animals were swabbed on 2 successive days with a cotton-tipped applicator saturated with 0.1 ml of fresh inoculum (31).
Quantification of C. albicans cells.
Establishment of C. albicans infection was evaluated by swabbing the inoculated oral cavity with a sterile cotton applicator, followed by plating on YEPD agar (25, 31). Samples were collected 2 days after inoculation and at the end of experiment. The cotton applicator was immediately immersed in 0.99 ml of sterile isotonic saline to obtain a dilution of 10−2, and it was agitated for 2 min. This dilution was considered to be 10−2. Dilutions up to 10−5 (0.1 ml) were cultured in duplicate in Sabouraud's dextrose agar at 37°C for 48 h. Candida colonies were counted in plates exhibiting between 30 and 300 colonies. Plates with less than 30 colonies in the 10−2 dilution were considered to have 101 cells (25).
Clinical lesions.
At the end of the experimental period, all animals were sacrificed by asphyxiation in a CO2 atmosphere and were then decapitated. The dorsal tongue was photographed in situ at a magnification of ×10 (3). Clinical lesions were measured with a digital imaging system (Técnicas Médicas MAB, Barcelona, Spain) and expressed as the percentage of the surface area of the tongue (percent area) that was covered with the lesions.
Tissue handling.
The tongues from the rats were hemidissected in the sagittal plane, with half of the lesion immersed in 10% buffered formalin for routine processing and the other half placed in 2.5% glutaraldehyde with 0.1 M Sorensen's phosphate buffer at 4°C (3).
Light microscopy.
Both hematoxylin and eosin and periodic acid-Schiff stains were used. C. albicans infection was assessed with a digital imaging system according to evidence of lesions and hyphal colonization on the dorsal tongue (3, 33). Tissue injury was determined by quantification of the number and type (normal, atrophic, and hypertropic) of papillae per microscopic field (magnification, ×46). A semiquantitative scale was devised to assess the degree of colonization of the epithelium by fungal hyphae. In this scale, the absence of colonization was given a score of 0, while maximal colonization, in excess of 50 hyphae, could be seen in each high-power field (magnification, ×400) was assigned a score of 4. The scores given were 1 for 1 to 5 hyphae, 2 for 6 to 15 hyphae, and 3 for 16 to 50 hyphae. The specimens were examined by one of us, who was blind as to the source. Three high-power fields per sample were examined for the light microscopy experiments.
Scanning electron microscopy preparation.
Following fixation for 24 h, the tissue was rinsed three times in buffer and postfixed in 1% phosphate-buffered osmium tetroxide (pH 7.4) for 1 h. After two buffer rinses, the specimens were dehydrated in ascending concentrations of ethanol, followed by critical point dehydration in a Denton DCP-1 critical point drying apparatus with liquid CO2. The tissue samples were affixed on aluminum stubs with silver conductive paint and were sputter-coated with gold-palladium by using a Hummer VI sputter-coating apparatus (Anatech Electronics, Garfield, N.J.). Specimens were viewed with a Zeiss 910 electron microscope (Zeiss, Oberkochen, Germany) operated at 20 kV (2).
Statistical analysis.
Statistical analysis of the quantitation of C. albicans cells in oral tissue was performed by one-way analysis of variance, followed by Bonferroni's t test. The percentage of areas of clinical lesions was analyzed by Student's t test (25). The Wilcoxon signed-rank sum test for paired comparisons and the Kruskal-Wallis test for multiple comparisons were used to determine the degree of colonization of the epithelium by fungal hyphae (33). Differences were considered significant at P < 0.05.
RESULTS
C. albicans counts at 2 and 15 days after inoculation (Table 1), as well as the percent area of clinical lesions in the dorsal tongue (Table 2), were increased in stressed rats compared with those in unstressed animals (differences, P < class="fig-table-link fig figpopup" style="TEXT-DECORATION: none" onclick="startTarget(this, 'figure', 1024, 800)" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=120028&rendertype=figure&id=f1" hoverintent_t="undefined" jquery1255197605890="2">(Fig.1)1) were observed in stressed animals (differences, P < class="fig-table-link fig figpopup" style="TEXT-DECORATION: none" onclick="startTarget(this, 'figure', 1024, 800)" href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=120028&rendertype=figure&id=f2" jquery1255197605890="3">(Fig.2)2) scored higher than untreated controls (differences, P <> 0.05), with the only exception that placebo increased the degree of colonization of the epithelium in unstressed animals. In contrast, treatment with alprazolam significantly (P < 0.05) reversed the adverse effects of stress in all parameters assayed.
Clinically evident lesions and inflammatory changes of the underlying connective tissue were observed 15 days after C. albicans inoculation. The latter were found in all experimental groups, but they were more evident in stressed rats. Animals showed macroscopic focal patchy atrophy of the dorsal tongue papillae. Light microscopy showed localized dense zones of hyphal penetration of the keratin layer in the giant conical papillae and filiform papillae of the dorsal tongue. Microabscesses in the keratin and the superficial spinous layers were observed in association with hyphal invasion. The underlying connective tissue showed a mild chronic inflammatory cell infiltrate. Those papillae that supported the Candida growth appeared shorter and blunter than the surrounding uninfected papillae.
Scanning electron microscopy of the dorsal tongues showed a higher loss of papillae in the giant conical and filiform areas of the specimens together with an increase in the size of the flat central portion of the lesion in stressed rats in comparison with unstressed animals. This adverse effect of stress was also reduced by the administration of alprazolam.
DISCUSSION
Our results show that stress exacerbates C. albicans infection of the tongues of rats. Significant increases in Candida counts, the percent area of clinical lesions, the percent abnormal papillae, and the colonization of the epithelium by fungal hyphae were found in stressed rats compared with those found in unstressed animals. Treatment with alprazolam partially reversed those adverse effects of stress on the development of oral candidiasis. Alprazolam was found to reverse many of the effects of stress on C. albicans infection of the tongues of rats, including Candida counts, the percent area of clinical lesions, the percent abnormal papillae, and the colonization of the epithelium by fungal hyphae.
Clinical and experimental observations indicate that the opportunistic proclivities of this fungus vary considerably, depending on the nature of the immunological defect of the victim.
Patients with qualitative or quantitative defects of phagocytes are mainly prone to the invasive form of this mycosis (10, 11, 37).
In contrast, defective T-cell-mediated immunity has been specifically associated with thrush and other forms of candidiasis limited to mucocutaneous surfaces (11, 12, 24, 26). Krause and Schaffner (28) demonstrated that cyclosporine, a relatively selective suppressor of T-cell-mediated immunity and NK cell activity, promoted the formation of thrushlike lesions on cyst surfaces and impeded the elimination of C. albicans from such lesions, but it had no effect on systemic candidiasis induced by intravenous inoculation.
Our results are in line with the previous literature on the stress-induced modulation of the immune system. Changes in murine splenic cytotoxic activities, mediated by NK cells and cytotoxic T lymphocytes have been reported (10-12, 24, 26, 32, 37). Stress also interferes with the activity of phagocytosis and T-cell-dependent antibody responses (21, 28).
The mechanism by which stress inhibits the cellular immune response has been widely studied. A molecular basis for bidirectional communication between the immune and neuroendocrine systems has been described previously (5). Cell-to-cell communication between the immune and the neuroendocrine systems is primarily mediated by hormones and neuropeptides that reach lymphoid organs and cells through the vascular system or directly through the autonomic connections between nerve endings and lymphoid organs (1, 8). Receptor sites are present in lymphoid cells for many hormones and neurotransmitters (6, 39). A number of molecules produced by cells of the nervous system such as ACTH, PRL, opioid peptides, GH, TSH, dynorphin, dopamine, and others have been shown to have the ability to modulate immune functions.
On the other hand, humoral factors generated by the immune system, such as thymic peptides and lymphokines, modulate neuroendocrine functions. In addition, in the course of lymphocyte activation, lymphoid cells may produce hormonal substances identical to those produced by the hypophysis, such as ACTH, TSH, GH, PRL, gonadotrophin, and β-endorphin (6).
At least one of the neuroendocrine responses to stress, such as the rise in plasma corticosterone concentrations via ACTH secretion, has an easily demonstrable destructive effect on specific cells and tissues that are required for optimal immune defense (4, 34). In our previous studies, we observed a stress-induced increase in ACTH levels proportional to the decrease in T-cell populations (15). Nevertheless, in these studies, we observed that adrenalectomized mice showed a lower pattern of immunosuppression in comparison with sham-operated mice. So, this led us to believe that other neuropeptides and neurotransmitters could be involved in the immunosuppressive response to stress.
The effects of alprazolam, an anxiolytic drug with high affinity for central BZD receptors on the pathogenicity of this opportunistic fungus could be attributed, at least in part, to its well-known protective effects against the immunosuppressive response to the type of stress assayed here. The recovery of the immune state of the victim could decrease the pathogenicity of this opportunistic fungus. In this regard, in our previous studies, we demonstrated that alprazolam reversed the suppressive effects of stress on the activity of phagocytosis, T-cell populations, the blastogenic response of spleen cells, murine splenic cytotoxic activities, mediated by NK cells and CTL. Fride et al. (23) found that low doses (0.02 to 1.0 mg/kg) of alprazolam significantly increased the NK cell activity, mixed leukocyte reactivity, and mitogen-induced lymphocyte proliferation in unstressed mice.
The mechanism of action of BZDs on the immune system remains to be defined. A dual approach has been described at the present time. First, central pharmacological effects related to the central type BZD receptors that facilitate inhibitory GABA neurotransmission in the central nervous system may regulate the release of neuroendocrine hormones involved in the immune response to stress. The ability of alprazolam to decrease the stress-induced increase of ACTH levels (29) has been demonstrated to play a important role in the immunoprotective effects of this drug. Nevertheless, significant immunoenhancing effects of alprazolam were also appreciated in stressed adrenalectomized rats (15), suggesting that the modulatory effect of this BZD agonist on other neurohormones like opioid peptides, PRL, melatonin, TSH, or GH could also be involved (13).
A second aspect of the effects of BZDs is the existence of a BZD receptor with high affinity on immune cells that express the so-called peripheral specificity for BZDs (41). Nevertheless, alprazolam is described in the literature as strict central type ligand of the BZD receptor (29). Alprazolam has potent PAF antagonist properties (27) that seem to affect T-cell, B-cell, and macrophage responses under in vitro conditions (9).
One could ask whether secondary (nonimmune or biochemical) effects of the drug treatment might account for the final observations and whether or not stress might break down the state of tolerance normally associated with Candida infection (as opposed to acting solely as an immunopotentiator). Although these considerations should be taken into account, our previous data concerning the immunomodulatory effects of alprazolam under stress conditions (14, 15, 18, 21, 38) lead us to consider immune changes as the main factor involved on the effects of stress and alprazolam on the evolution of oral candidiasis in rats.
A second question concerns the biological significance of our results. Although our data at present show stress may leave the subject vulnerable to the action of C. albicans and provide evidence of a protective effect of alprazolam on the development of oral candidiasis in rats, the biological significance and health relatedness of these findings should be assessed. In this respect, differences between untreated stressed rats and placebo- or alprazolam-stressed rats are statistically significant, but in some parameters, they are not striking. Moreover, there is a relationship between differences obtained in different determinations, but there is not a mathematical correlation as expected.
The large number of interactions at molecular, cellular, and functional levels between the nervous system and the immune system characterizing the operational compositions and expressions of the neuroimmune network make complex isolation of the pathways in which stress and alprazolam may be involved in the regulation of the host defense mechanisms against infection. Nevertheless, the literature has provided evidence that stress-induced immunosuppression and alprazolam-induced immunoprotection are in a relationship with susceptibility to bacteria (16), virus (20), and, as a conclusion of the present investigation, Candida infection.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=120028
The Impact of Sleep Deprivation on Hormones and Metabolism
Sleep is the main stressor on our bodies. Sleep curtailment leads to majour disorders of our endocrine system, which, in turn, weaken our bodies' ability to fight infection in particular and disease in general.
Cancer, diabetes, heart disease and many other severe maladies are caused in more cases that it is popularly believed, by stress and sleep deprivation.
I will start with an educational article which, in a few words, explains the harmful mechanism that we expose our body to when sleep deprived.
-Vera
The Impact of Sleep Deprivation on Hormones and Metabolism
Eve Van Cauter, PhD; Kristen Knutson, PhD; Rachel Leproult, PhD; Karine Spiegel, PhD
Introduction
Sleep loss can occur as a result of habitual behavior or due to the presence of a pathological condition that is associated with reduced total sleep time. This column focuses on the impact of behavioral sleep curtailment, an endemic condition in modern society, and provides evidence against the old notion that "sleep is for the mind, and not for the rest of the body."
Prevalence of Sleep Curtailment in Modern Society
Sleep curtailment is a hallmark of modern society, one that is often considered harmless and efficient. The advent of artificial light has permitted the curtailment of sleep to the minimum tolerable and an increase in the time available for work and leisure. In our 24-hour-a-day society, millions work during the night and sleep during the day, a schedule that generally results in substantial sleep loss.
Figure 1 illustrates changes in self-reported sleep duration over the past 50 years. In 1960, a survey of over 1 million people found a modal sleep duration of 8.0-8.9 hours.[1] In 2000, 2001, and 2002, polls conducted by the National Sleep Foundation indicated that the average duration of sleep for Americans had fallen to 6.9-7.0 hours.[2] Overall, sleep duration thus appears to have decreased by 1.5-2 hours during the second half of the 20th century. Today, many people are in bed only 5-6 hours per night on a regular basis.
The 2 major pathways by which sleep affects the release of hormones are the hypothalamic-pituitary axes and the autonomous nervous system.
The release of hormones by the pituitary — the "master" endocrine organ that controls the secretion of other hormones from the peripheral endocrine glands — is markedly influenced by sleep. Modulation of pituitary-dependent hormonal release is partly mediated by the modulation of the activity of hypothalamic-releasing and/or hypothalamic-inhibiting factors controlling pituitary function. During sleep, these hypothalamic factors may be activated — as in the case of growth hormone (GH)-releasing hormone — or inhibited, as is the case for corticotropin-releasing hormone.
The other pathway by which sleep affects peripheral endocrine regulation is via the modulation of autonomic nervous system activity. During deep sleep, sympathetic nervous system activity is generally decreased and parasympathetic nervous system activity is increased. Sleep loss is associated with an elevation of sympathovagal balance, with higher sympathetic but lower parasympathetic tone. Most endocrine organs are sensitive to changes in sympathovagal balance. Well-documented examples are pancreatic insulin secretion and release by the fat cells of leptin, an appetite-suppressing hormone.
A profound and generalized impact of sleep loss on the endocrine system should therefore be expected. Until recently, however, it was considered unlikely that the adverse effects of sleep deprivation on endocrine function would be long-term. The studies from which this notion was drawn examined the effects of only 1 or 2 nights of acute total sleep deprivation. In general, the data suggested that endocrine alterations that occurred during the sleepless night(s) were completely reversed during recovery sleep.
More recently, a few studies have examined the impact on hormones, metabolism, and immune function of the much more common, real-life situation — chronic partial sleep deprivation.[3-5] The earliest study measured hormonal and metabolic parameters in subjects studied after 6 days of sleep restriction (4-hour bedtime) and after full sleep recovery (6 days of 12-hour bedtime).[3] Subsequent studies examined the impact of less severe sleep restriction (6.5 hours per night) over 1 week[4] as well as the effects of short-term sleep curtailment (2 days with 4-hour vs 12-hour bedtime).[5]
Alterations of Pituitary-Dependent Hormones During Sleep Loss
The first effect of partial sleep loss on circulating levels of pituitary-dependent hormones to be documented under various study conditions is an increase in the early evening levels of the stress hormone cortisol.[3,6] Normally at that time of day, cortisol concentrations are rapidly decreasing to attain minimal levels shortly before habitual bedtime.
The rate of decrease of cortisol concentrations in the early evening was approximately 6-fold slower in subjects who had undergone 6 days of sleep restriction than in subjects who were fully rested.[3] Elevations of evening cortisol levels in chronic sleep loss are likely to promote the development of insulin resistance, a risk factor for obesity and diabetes.
The upper and middle panels of Figure 2 illustrate the impact of sleep restriction on the thyroid axis.[3] After 6 days of 4-hour sleep time, the normal nocturnal thyroid-stimulating hormone (TSH) rise was strikingly decreased, and the overall mean TSH levels were reduced by more than 30%.[3] A normal pattern of TSH release reappeared when the subjects had fully recovered. Differences in TSH profiles between the 2 bedtime conditions were probably related to changes in thyroid hormone concentrations via a negative-feedback regulation, because the free thyroxine index (FT4I) was higher in the sleep-restriction condition than in the fully rested condition (middle panels of Figure 2). Thyroid axis function was thus markedly altered by partial recurrent sleep restriction.
Figure 2.
Levels of thyroid-stimulating hormone (TSH), free thyroxine index, and leptin in sleep-deprived vs well-rested subjects. From top to bottom, 24-hour (+SEM) profiles of TSH, free thyroxine indexes, and leptin in healthy young subjects when submitted to partial sleep restriction for 6 days (4-hour sleep times; mean total sleep time during previous 2 nights, 3 hours 49 minutes; left panels) and after full sleep recovery (12-hour sleep times for 6 nights; mean total sleep time during previous 2 nights, 9 hours 3 minutes; right panels). The black bars represent the sleep periods.[3,10]
The temporal organization of GH secretion is also altered by chronic partial sleep loss.[7] The normal single GH pulse occurring shortly after sleep onset splits into 2 smaller pulses, 1 before sleep and 1 after sleep; as a result, the peripheral tissues are exposed to high GH levels for an extended period of time, which, because GH has anti-insulin-like effects, could also have an adverse impact on glucose tolerance.
Impact of Sleep Loss on Hormones Controlling Appetite
Sleeping and feeding are intricately related. Animals faced with food shortage or starvation sleep less;[8] conversely, animals subjected to total sleep deprivation for prolonged periods of time increase their food intake markedly.[9] Recent studies in humans have shown that the levels of hormones that regulate appetite are profoundly influenced by sleep duration. Sleep loss is associated with an increase in appetite that is excessive in relation to the caloric demands of extended wakefulness.
The regulation of leptin, a hormone released by the fat cells that signals satiety to the brain and thus suppresses appetite, is markedly dependent on sleep duration. After 6 days of bedtime restriction to 4 hours per night, the plasma concentration of leptin was markedly decreased, particularly during the nighttime.[10] The magnitude of this decrease was comparable to that occurring after 3 days of restricting caloric intake by approximately 900 kcal/day. But the subjects in the sleep-restriction condition received identical amounts of caloric intake and had similar levels of physical activity as when they were fully rested. Thus, leptin levels were signaling a state of famine in the midst of plenty.
In a later study, the levels of ghrelin, a peptide that is secreted by the stomach and stimulates appetite, were measured with the levels of leptin after 2 days of sleep restriction (4 hours of sleep) or sleep extension (10 hours of bedtime).[5] The subjects also assessed their levels of hunger and appetite at regular intervals. Sleep restriction was associated with reductions in leptin (the appetite suppressant) and elevations in ghrelin (the appetite stimulant) and increased hunger and appetite, especially an appetite for foods with high-carbohydrate contents. Similar findings were obtained simultaneously in a large epidemiologic study in which sleep duration and morning levels of leptin and ghrelin were measured in over 1,000 subjects.[11] The Table summarizes the remarkable concordance between the results of the 2 studies. Despite the differences in study design, both studies found a decrease in the satiety hormone leptin and an increase in appetite-stimulating ghrelin with short sleep.
Sleep loss therefore seems to alter the ability of leptin and ghrelin to accurately signal caloric need and could lead to excessive caloric intake when food is freely available. The findings also suggest that compliance with a weight-loss diet involving caloric restriction may be adversely affected by sleep restriction.
During the second half of the 20th century, the incidence of obesity has nearly doubled, and this trend is a mirror image of the decrease in self-reported sleep duration illustrated in Figure 1. The discovery of a profound alteration in the neuroendocrine control of appetite in conditions of sleep loss is consistent with the conclusions of several epidemiologic studies that revealed a negative association between self-reported sleep duration and body mass index. Taken together, the current evidence suggests a possible role for chronic sleep loss in the current epidemic of obesity.
Metabolic Implications of Recurrent Sleep Curtailment
Recent work also indicates that sleep loss may adversely affect glucose tolerance and involve an increased risk of type 2 diabetes.
In young, healthy subjects who were studied after 6 days of sleep restriction (4 hours in bed) and after full sleep recovery, the levels of blood glucose after breakfast were higher in the state of sleep debt despite normal or even slightly elevated insulin responses.[3] The difference in peak glucose levels in response to breakfast averaged ±15 mg/dL, a difference large enough to suggest a clinically significant impairment of glucose tolerance.
These findings were confirmed by the results of intravenous glucose tolerance testing.[3] Indeed, the rate of disappearance of glucose post injection — a quantitative measure of glucose tolerance — was nearly 40% slower in the sleep-debt condition than after recovery, and the acute insulin response to glucose was reduced by 30%. Glucose tolerance measured at the end of the recovery period was similar to that reported in an independent study[12] in young, healthy men, but glucose tolerance in the state of sleep debt was comparable to that reported for older adults with impaired glucose tolerance.[13]
Thus, less than 1 week of sleep restriction can result in a prediabetic state in young, healthy subjects. Of note, the adverse impact of sleep deprivation on glucose tolerance demonstrated in laboratory studies is consistent with the finding of an increased risk of symptomatic diabetes with short sleep in a cohort study of women.[14]
Multiple mechanisms are likely to mediate the adverse effects of sleep curtailment on parameters of glucose tolerance, including decreased cerebral glucose utilization, increases in sympathetic nervous system activity, and abnormalities in the pattern of release of the counterregulatory hormones cortisol and GH.
Conclusion
Clearly, sleep is not only for the brain but also for the rest of the body. Recent evidence suggests that sleep loss, a highly prevalent — and often strongly encouraged — condition in modern society could be a risk factor for major chronic diseases, including obesity and diabetes.
Supported by an independent educational grant from Pfizer/Neurocrine.
http://cme.medscape.com/viewarticle/502825
Cancer, diabetes, heart disease and many other severe maladies are caused in more cases that it is popularly believed, by stress and sleep deprivation.
I will start with an educational article which, in a few words, explains the harmful mechanism that we expose our body to when sleep deprived.
-Vera
The Impact of Sleep Deprivation on Hormones and Metabolism
Eve Van Cauter, PhD; Kristen Knutson, PhD; Rachel Leproult, PhD; Karine Spiegel, PhD
Introduction
Sleep loss can occur as a result of habitual behavior or due to the presence of a pathological condition that is associated with reduced total sleep time. This column focuses on the impact of behavioral sleep curtailment, an endemic condition in modern society, and provides evidence against the old notion that "sleep is for the mind, and not for the rest of the body."
Prevalence of Sleep Curtailment in Modern Society
Sleep curtailment is a hallmark of modern society, one that is often considered harmless and efficient. The advent of artificial light has permitted the curtailment of sleep to the minimum tolerable and an increase in the time available for work and leisure. In our 24-hour-a-day society, millions work during the night and sleep during the day, a schedule that generally results in substantial sleep loss.
Figure 1 illustrates changes in self-reported sleep duration over the past 50 years. In 1960, a survey of over 1 million people found a modal sleep duration of 8.0-8.9 hours.[1] In 2000, 2001, and 2002, polls conducted by the National Sleep Foundation indicated that the average duration of sleep for Americans had fallen to 6.9-7.0 hours.[2] Overall, sleep duration thus appears to have decreased by 1.5-2 hours during the second half of the 20th century. Today, many people are in bed only 5-6 hours per night on a regular basis.
The 2 major pathways by which sleep affects the release of hormones are the hypothalamic-pituitary axes and the autonomous nervous system.
The release of hormones by the pituitary — the "master" endocrine organ that controls the secretion of other hormones from the peripheral endocrine glands — is markedly influenced by sleep. Modulation of pituitary-dependent hormonal release is partly mediated by the modulation of the activity of hypothalamic-releasing and/or hypothalamic-inhibiting factors controlling pituitary function. During sleep, these hypothalamic factors may be activated — as in the case of growth hormone (GH)-releasing hormone — or inhibited, as is the case for corticotropin-releasing hormone.
The other pathway by which sleep affects peripheral endocrine regulation is via the modulation of autonomic nervous system activity. During deep sleep, sympathetic nervous system activity is generally decreased and parasympathetic nervous system activity is increased. Sleep loss is associated with an elevation of sympathovagal balance, with higher sympathetic but lower parasympathetic tone. Most endocrine organs are sensitive to changes in sympathovagal balance. Well-documented examples are pancreatic insulin secretion and release by the fat cells of leptin, an appetite-suppressing hormone.
A profound and generalized impact of sleep loss on the endocrine system should therefore be expected. Until recently, however, it was considered unlikely that the adverse effects of sleep deprivation on endocrine function would be long-term. The studies from which this notion was drawn examined the effects of only 1 or 2 nights of acute total sleep deprivation. In general, the data suggested that endocrine alterations that occurred during the sleepless night(s) were completely reversed during recovery sleep.
More recently, a few studies have examined the impact on hormones, metabolism, and immune function of the much more common, real-life situation — chronic partial sleep deprivation.[3-5] The earliest study measured hormonal and metabolic parameters in subjects studied after 6 days of sleep restriction (4-hour bedtime) and after full sleep recovery (6 days of 12-hour bedtime).[3] Subsequent studies examined the impact of less severe sleep restriction (6.5 hours per night) over 1 week[4] as well as the effects of short-term sleep curtailment (2 days with 4-hour vs 12-hour bedtime).[5]
Alterations of Pituitary-Dependent Hormones During Sleep Loss
The first effect of partial sleep loss on circulating levels of pituitary-dependent hormones to be documented under various study conditions is an increase in the early evening levels of the stress hormone cortisol.[3,6] Normally at that time of day, cortisol concentrations are rapidly decreasing to attain minimal levels shortly before habitual bedtime.
The rate of decrease of cortisol concentrations in the early evening was approximately 6-fold slower in subjects who had undergone 6 days of sleep restriction than in subjects who were fully rested.[3] Elevations of evening cortisol levels in chronic sleep loss are likely to promote the development of insulin resistance, a risk factor for obesity and diabetes.
The upper and middle panels of Figure 2 illustrate the impact of sleep restriction on the thyroid axis.[3] After 6 days of 4-hour sleep time, the normal nocturnal thyroid-stimulating hormone (TSH) rise was strikingly decreased, and the overall mean TSH levels were reduced by more than 30%.[3] A normal pattern of TSH release reappeared when the subjects had fully recovered. Differences in TSH profiles between the 2 bedtime conditions were probably related to changes in thyroid hormone concentrations via a negative-feedback regulation, because the free thyroxine index (FT4I) was higher in the sleep-restriction condition than in the fully rested condition (middle panels of Figure 2). Thyroid axis function was thus markedly altered by partial recurrent sleep restriction.
Figure 2.
Levels of thyroid-stimulating hormone (TSH), free thyroxine index, and leptin in sleep-deprived vs well-rested subjects. From top to bottom, 24-hour (+SEM) profiles of TSH, free thyroxine indexes, and leptin in healthy young subjects when submitted to partial sleep restriction for 6 days (4-hour sleep times; mean total sleep time during previous 2 nights, 3 hours 49 minutes; left panels) and after full sleep recovery (12-hour sleep times for 6 nights; mean total sleep time during previous 2 nights, 9 hours 3 minutes; right panels). The black bars represent the sleep periods.[3,10]
The temporal organization of GH secretion is also altered by chronic partial sleep loss.[7] The normal single GH pulse occurring shortly after sleep onset splits into 2 smaller pulses, 1 before sleep and 1 after sleep; as a result, the peripheral tissues are exposed to high GH levels for an extended period of time, which, because GH has anti-insulin-like effects, could also have an adverse impact on glucose tolerance.
Impact of Sleep Loss on Hormones Controlling Appetite
Sleeping and feeding are intricately related. Animals faced with food shortage or starvation sleep less;[8] conversely, animals subjected to total sleep deprivation for prolonged periods of time increase their food intake markedly.[9] Recent studies in humans have shown that the levels of hormones that regulate appetite are profoundly influenced by sleep duration. Sleep loss is associated with an increase in appetite that is excessive in relation to the caloric demands of extended wakefulness.
The regulation of leptin, a hormone released by the fat cells that signals satiety to the brain and thus suppresses appetite, is markedly dependent on sleep duration. After 6 days of bedtime restriction to 4 hours per night, the plasma concentration of leptin was markedly decreased, particularly during the nighttime.[10] The magnitude of this decrease was comparable to that occurring after 3 days of restricting caloric intake by approximately 900 kcal/day. But the subjects in the sleep-restriction condition received identical amounts of caloric intake and had similar levels of physical activity as when they were fully rested. Thus, leptin levels were signaling a state of famine in the midst of plenty.
In a later study, the levels of ghrelin, a peptide that is secreted by the stomach and stimulates appetite, were measured with the levels of leptin after 2 days of sleep restriction (4 hours of sleep) or sleep extension (10 hours of bedtime).[5] The subjects also assessed their levels of hunger and appetite at regular intervals. Sleep restriction was associated with reductions in leptin (the appetite suppressant) and elevations in ghrelin (the appetite stimulant) and increased hunger and appetite, especially an appetite for foods with high-carbohydrate contents. Similar findings were obtained simultaneously in a large epidemiologic study in which sleep duration and morning levels of leptin and ghrelin were measured in over 1,000 subjects.[11] The Table summarizes the remarkable concordance between the results of the 2 studies. Despite the differences in study design, both studies found a decrease in the satiety hormone leptin and an increase in appetite-stimulating ghrelin with short sleep.
Sleep loss therefore seems to alter the ability of leptin and ghrelin to accurately signal caloric need and could lead to excessive caloric intake when food is freely available. The findings also suggest that compliance with a weight-loss diet involving caloric restriction may be adversely affected by sleep restriction.
During the second half of the 20th century, the incidence of obesity has nearly doubled, and this trend is a mirror image of the decrease in self-reported sleep duration illustrated in Figure 1. The discovery of a profound alteration in the neuroendocrine control of appetite in conditions of sleep loss is consistent with the conclusions of several epidemiologic studies that revealed a negative association between self-reported sleep duration and body mass index. Taken together, the current evidence suggests a possible role for chronic sleep loss in the current epidemic of obesity.
Metabolic Implications of Recurrent Sleep Curtailment
Recent work also indicates that sleep loss may adversely affect glucose tolerance and involve an increased risk of type 2 diabetes.
In young, healthy subjects who were studied after 6 days of sleep restriction (4 hours in bed) and after full sleep recovery, the levels of blood glucose after breakfast were higher in the state of sleep debt despite normal or even slightly elevated insulin responses.[3] The difference in peak glucose levels in response to breakfast averaged ±15 mg/dL, a difference large enough to suggest a clinically significant impairment of glucose tolerance.
These findings were confirmed by the results of intravenous glucose tolerance testing.[3] Indeed, the rate of disappearance of glucose post injection — a quantitative measure of glucose tolerance — was nearly 40% slower in the sleep-debt condition than after recovery, and the acute insulin response to glucose was reduced by 30%. Glucose tolerance measured at the end of the recovery period was similar to that reported in an independent study[12] in young, healthy men, but glucose tolerance in the state of sleep debt was comparable to that reported for older adults with impaired glucose tolerance.[13]
Thus, less than 1 week of sleep restriction can result in a prediabetic state in young, healthy subjects. Of note, the adverse impact of sleep deprivation on glucose tolerance demonstrated in laboratory studies is consistent with the finding of an increased risk of symptomatic diabetes with short sleep in a cohort study of women.[14]
Multiple mechanisms are likely to mediate the adverse effects of sleep curtailment on parameters of glucose tolerance, including decreased cerebral glucose utilization, increases in sympathetic nervous system activity, and abnormalities in the pattern of release of the counterregulatory hormones cortisol and GH.
Conclusion
Clearly, sleep is not only for the brain but also for the rest of the body. Recent evidence suggests that sleep loss, a highly prevalent — and often strongly encouraged — condition in modern society could be a risk factor for major chronic diseases, including obesity and diabetes.
Supported by an independent educational grant from Pfizer/Neurocrine.
http://cme.medscape.com/viewarticle/502825
TABLE OF CONTENTS
1. The Impact of Sleep Deprivation on Hormones and Metabolism
2. Effects of Psychological Stress and Alprazolam on Development of Oral Candidiasis in Rats
3. CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production
4. Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird
5. Endogenous substance P inhibits the expression of corticotropin-releasing hormone during a chronic inflammatory stress
6. Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides
7. Tetrahydroprogesterone Attenuates the Endocrine Response to Stress
8. Tetrahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety and alters the release of corticotropin-releasing hormone
9. Sleep deprivation effects on the activity of the hypothalamic–pituitary–adrenal and growth axes: potential clinical implications
10. Immobilisation stress induces a paradoxical sleep rebound in rat
11. STRESS-INDUCED SLEEP DEPRIVATION MODIFIES CORTICOTROPIN RELEASING FACTOR (CRF) LEVELS
12. Protective effect of alprazolam against sleep deprivation-induced behavior alterations and oxidative damage in mice
13. Increased susceptibility to disseminated Candidiasis in interleukin-6 deficient mice
14. CRH and GPI-anchored Gas families are FUNGAL ANTIGENS
15. Involvement of the corticotropin-releasing hormone system in the pathogenesis of acne vulgaris
16. CRH Enhances Retention of a Spatial Memory
17. Corticotropin-Releasing Hormone (CRH) Downregulates the Function of Its Receptor (CRF1)
2. Effects of Psychological Stress and Alprazolam on Development of Oral Candidiasis in Rats
3. CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production
4. Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird
5. Endogenous substance P inhibits the expression of corticotropin-releasing hormone during a chronic inflammatory stress
6. Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides
7. Tetrahydroprogesterone Attenuates the Endocrine Response to Stress
8. Tetrahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety and alters the release of corticotropin-releasing hormone
9. Sleep deprivation effects on the activity of the hypothalamic–pituitary–adrenal and growth axes: potential clinical implications
10. Immobilisation stress induces a paradoxical sleep rebound in rat
11. STRESS-INDUCED SLEEP DEPRIVATION MODIFIES CORTICOTROPIN RELEASING FACTOR (CRF) LEVELS
12. Protective effect of alprazolam against sleep deprivation-induced behavior alterations and oxidative damage in mice
13. Increased susceptibility to disseminated Candidiasis in interleukin-6 deficient mice
14. CRH and GPI-anchored Gas families are FUNGAL ANTIGENS
15. Involvement of the corticotropin-releasing hormone system in the pathogenesis of acne vulgaris
16. CRH Enhances Retention of a Spatial Memory
17. Corticotropin-Releasing Hormone (CRH) Downregulates the Function of Its Receptor (CRF1)
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