Dissertationen zum Thema „Melatonin Physiological effect“

Wound healing is a dynamic process that ultimately leads to restoration of tissue integrity and function. The pineal gland hormone melatonin is known for its anti-oncotic, anti- inflammatory and immuno-modulatory effects. However, its role in wound healing has not been established. Since melatonin is synthesised endogenously, we primarily sought to investigate whether the melatonin receptors played a role in the wound healing process. Using immunohistochemical methods and Western blot analysis we observed that MT₁ was normally absent in the rat skin but was strongly expressed on day 1 to day 3 post wounding in the epidermis adjacent to the wound edge. MT₁ expression was restricted to the stratum granulosum and stratum spinosum layers of the epidermis in the rat wounds. MT₁ expression declined thereafter and became nonexistent by day 21 when the wound had completely healed. In contrast, MT₂ was constitutively expressed in all the layers of the normal rat epidermis. MT₂ expression gradually decreased at the injury site following wounding but returned to the normal profile by day 21. Aged rat epidermis showed similar MT₁ and MT₂ expression as adult rats. The profile of tissue distribution of MT₁ and MT₂ in the human epidermis was comparable to the rat epidermis. In the CVUs MT₁ and MT₂ localisation profiles resembled that of a healing wound, akin to a day 1 or day 3 rat dermal wound, during the inflammatory phase. Surprisingly, in contrast to all the tissues investigated, MT₁ was also localised in the stratum basale layer of the keloid epidermis. MT₂ localisation in the same keloid tissues however resembled normal human skin profiles. Secondly, we determined the effects of exogenously administered melatonin, on scarring and wound healing, using a full thickness incisional model of wound healing in rats. Melatonin treatment significantly improved the quality of scarring by day 21. However, our findings would have been strengthened by a more explicit wound closure analysis, measurement of granulation tissue weight, tensile strength, hydroxyl proline content and immunohistochemical assessments of neutrophil infiltration, macrophages, fibroblasts, myofibroblasts and reepithelialization. The treatment also accelerated the angiogenic process and enhanced the VEGF protein profile. Arginase generates proline, the building block for collagen synthesis. Melatonin treatment increased arginase activity and consequently would increase collagen synthesis from day 1. An increase in NOS activity and therefore NO production is known to be detrimental during inflammation. However, various studies have also shown that the NO is essential for granulation tissue formation. Melatonin treatment significantly decreased iNOS activity during the acute inflammatory phase in this study, but significantly increased iNOS activity during the resolving phase. Other markers of inflammatory response and repair were also examined in this study. COX-2 has been shown to play an anti-inflammatory role and melatonin increased COX-2 activity and protein following wounding. SOD (the antioxidant enzyme) activity was also significantly increased during the chronic inflammatory phase on melatonin administration. HO-1 and HO-2 isoforms have also been previously demonstrated to participate in the repair process. Melatonin treatment increased up-regulation of both HO-1 and HO-2 protein expression in the wounded skin. A significant decrease in all the mitochondrial enzyme activities (except complex-II-III), was observed post wounding. Melatonin treatment restored the complex activities to near normal levels. Melatonin also protected mitochondrial membrane integrity and reduced oxidative stress as evidenced by the maintained level of aconitase and citrate synthase activities at near normal levels. In vitro experiments using macrophage and fibroblast cell lines illustrated that melatonin may decrease NOS activity and protein profiles indirectly by stimulating arginase activity and thereby depleting the availability of arginine. This study is the first to fully demonstrate the distribution of melatonin receptors in normal and abnormal wounds. Improvement in the quality of scarring in a rat model of wound healing on melatonin administration is promising but much more quantitative work and preclinical studies are required before melatonin advances into clinical assessment.

There is ongoing controversy about the possibility of adverse biological effects from environmental exposures to electric and magnetic fields. These fields are produced by all electrical equipment and appliances including electrical transmission lines. The objective of this environmental science study was to investigate the possible effects of a high voltage transmission line on domestic sheep (Ovis aries L,), a species that can often be found near such lines. The study was primarily designed to determine whether a specific effect of electric and magnetic fields found in laboratory animals also occurs in livestock under natural environmental conditions. The effect is the ability of fields, at levels found in the environment, to significantly depress the normally high nocturnal concentrations of the pineal hormone melatonin. Melatonin mediates the reproductive response to changes in photoperiod in seasonal breeders such as sheep. Factors which modify the production of nocturnal melatonin in sheep can have important effects on the timing of seasonal reproduction including the onset of puberty in this species. Ten female Suffolk lambs were penned for 10 months directly beneath a 500-kV transmission line near Estacada, Oregon. Ten other lambs of the same type were penned in a control area away from the transmission line where electric and magnetic fields were at ambient levels. Serum melatonin was analyzed by radioimmunoassay (RIA) from 6618 blood samples collected at 0.5 to 3-hour intervals over eight 48-hour periods. Serum progesterone was analyzed by RIA from blood samples collected twice weekly beginning when the lambs were 23 weeks old. This hormone was used to measure the onset of puberty. Serum cortisol was also assayed by RIA from the blood samples collected during the 48-hour samples. This was done to assess whether exposure to the transmission line produced stress in the growing lambs. Other supplemental biological data collected included body weight gain, wool growth, and behavior. An extensive study was conducted by engineers from the Bonneville Power Administration (BPA) to measure electric and magnetic fields and noise to which the lambs were exposed. This was accomplished by installing permanent monitors near both the control and line pens. Results showed that lambs in both the control and line groups had the typical pattern of melatonin secretion consisting of low daytime and high nighttime serum concentrations. There were no statistically significant differences between groups in melatonin levels, or in the phase or duration of the nighttime melatonin elevation. Age at puberty and number of reproductive cycles also did not differ between groups. Serum cortisol showed a circadian rhythm with highest concentrations during the day. Cortisol concentrations also seemed to reflect effects of known stressors on livestock, e.g., weaning, introduction to new housing, and vehicle transport. There were, however, no differences in cortisol concentrations between groups. Statistical analyses on other biological parameters revealed no differences between groups for body weight gain, wool growth, or behavior. The electrical monitoring program verified that the line group lambs were exposed to electric and magnetic fields at levels typical of those found beneath commercial 500-kV transmission lines. In summary, the large effect of electric and magnetic fields on melatonin concentrations reported in laboratory animals was not observed in this study of sheep.

"Life" may be characterized as a controlled stationary flow equilibrium, maintained by energy consuming chemical reactions. The physiological functioning of these life systems include at least 28 of the elements isolated on the periodic table thus far, most of which are metals. However, as with Paracelsus Principle: "The dose makes the poison", there exists a definite link between metal levels, essential and toxic, and the onset of neurodegenerative diseases. The economic costs of brain dysfunction are enormous, but this pales in comparison to the staggering emotional toll on the victims themselves and their families. In an attempt to improve the understanding of the causes of neurodegeneration, this study focuses on one potential aspect: the possible link between metals and neurotransmitter homeostasis utilising a variety of electronanalytical techniques. Adsorptive cathodic stripping voltammetry was employed to investigate the binding affinities and complex formation of melatonin and its precursor serotonin with calcium, potassium, sodium, lithium and aluminium. The results showed that all the metals studied formed complexes with both pineal indoleamines. However, the stability and affmity of the ligands toward the various metals varied greatly. The study suggests a further role for melatonin, that of metalloregulator and possible metal detoxifier in the brain, the in vivo studies which followed will further substantiate this notion. This research additionally focused on the cholinergic system, in particular acetylcholine complex formation studies with mercury, lead, cadmium, copper and zinc using the adsorptive cathodic stripping voltammetry method. The formation and characterisation of a solid mercury-acetylcholine complex lent further strength to the in situ electrochemical complex formation observed. The results showed the preference of acetylcholine for environmentally toxic heavy metals (such as Cd²⁺) over those divalent cations that occur naturally in the body. The possible metalloregulatory role melatonin played in the three brain regIOns: cerebellum, cortex and corpus striatum of male Wistar rats was studied as an in vivo extension of the earlier in vitro studies. Anodic stripping voltammetry was employed to detect metal levels present. The results showed that daily injections of melatonin was responsible for significantly decreasing copper(I), cadmium(II) and lead(II) levels in various regions of the rat brain of those animals that had undergone a pinealectomy in comparison to the saline injected group having undergone the same treatment. Histological and electrochemical stripping techniques were applied to investigate the implications of high A1³⁺ levels in the brain regions, particularly the hippocampus. Melatonin showed signs of promise in indirect symptom alleviation and by significantly decreasing A1³⁺ levels in rats that had been dosed with melatonin prior to A1³⁺ treatments in comparison with the control groups. Finally a preliminary study outlining a method for the production of a calcium selective microelectrode was undertaken. Further work is still needed to optimise the microelectrode production as well as its possible applications. However, whilst the overall conclusions of this entire multidisciplinary study may indeed only be in effect one piece of a very large puzzle on neurodegenerative diseases, this piece will no doubt serve as a building block for further ideas and work in this field.

Melatonin is a hormone secreted by the pineal gland, which is known to modulate biological rhythms in mammals. This study investigated the effect of food access schedule and dietary composition on serum melatonin and pineal NAT activity in adult male Wistar rats. These rats were maintained on a 12:12 h light:dark schedule with lights on at 0800h. The rats were randomly assigned to two dietary groups. A group was simultaneously fed a protein-rich and carbohydrate-rich granulated diet and the other group fed granulated rat chow. Each dietary group was further divided based on dietary feeding schedules. Animals were fed between 0800--1600 h or fed ad libitum. The study revealed that protein intake of rats fed the dietary choice was lower with the restricted access than in the free access. In rats fed dietary choice, the nocturnal melatonin levels and pineal NAT activity were significantly lower under the restricted access feeding when compared to the ad libitum feeding schedule. This was not observed in rats fed single chow diet. In conclusion our data demonstrate that food composition does affect the nocturnal synthesis of melatonin as well as the activity of the enzyme NAT. This could be via dietary intake of tryptophan, which is a precursor melatonin synthesis in the pineal gland.

Critical life history events such as breeding, migration and hibernation must take place in the correct environmental context to minimize deleterious consequences on survival and reproductive fitness. Neuroendocrine mechanisms synchronizing internal physiological states with extrinsic environmental cues are vital to timing life history events appropriately. Secretion of the pineal hormone melatonin is sensitive to light and temperature cues, which provides a physiological indicator of time of day and time of year for organisms. Melatonin influences seasonal reproduction in a variety of vertebrates, likely by altering the synthesis and/or release of reproductive neuropeptides in the brain. The neuropeptides arginine vasotocin and its mammalian homologue, arginine vasopressin, are well-known modulators of reproductive and sociosexual behavior across vertebrate taxa, and are likely targets of melatonin in the context of seasonal reproduction. There is extensive evidence that vasotocin/vasopressin innervation in the brain is subject to seasonal variation, and that this variation is frequently sexually dimorphic. However, evidence that melatonin directly modulates this important neuropeptide system is lacking. Melatonin receptor 1a (MT1 in mammals) may be responsible for mediating melatonin's influence on brain vasotocin, as it is known to regulate seasonal reproduction in a variety of vertebrates. In the present study, I asked whether melatonin influences brain vasotocin in male green treefrogs (Hyla cinerea), and compared the distribution of melatonin receptor 1a in the brain of green treefrogs between sexes and seasons. Adult male and female green treefrogs were collected from field sites in Louisiana during the summer breeding season. Summer animals were acclimated to lab conditions for 3 weeks, then euthanized and their brains collected. Winter animals were maintained in the lab for four months under incrementally changing photo-, thermo-, and hygroperiod regimes that mimicked the transition to winter in their natural habitat, followed by euthanasia and brain collection. A subset of winter males (Experiment 1) were implanted with melatonin-filled or blank silastic capsules for a period of one month prior to euthanasia and brain collection. Brains of these males were processed for vasotocin immunohistochemistry. I quantified AVT-ir cell number in Experiment 1 males in the nucleus accumbens (NAcc), amygdala and caudal striatum (AMG), preoptic area (POA), suprachaismatic nucleus (SCN), and ventral hypothalamus (VH). Melatonin did not influence brain vasotocin-ir cell number in any brain region. Brains from untreated summer and winter males and females were collected and processed for MT1 immunohistochemistry. MT1-ir cells were quantified in the NAcc, striatum (STR), AMG, POA, SCN, and VH. In all regions quantified, reproductively active males had significantly more MT1-ir cells than nonreproductive males. Within the summer breeding season, males had significantly more MT1-ir cells in the NAcc than did reproductively active females. In all other regions there was no significant difference in MT1-ir cell number between reproductively active males and females. Collectively, these data suggest that melatonin modulates vasotocin via MT1. These findings assist in elucidating the neuroendocrine mechanisms by which vertebrates integrate seasonal cues with physiology to correctly time critical life history events.

Dysfunction of central dopaminergic systems has been implicated in neuroendocrine, neurodegenerative and psychiatric disorders. Monoamine oxidase and catechol-Omethyltransferase represent the key catabolic enzymes of dopamine, terminating neurotransmission following synaptic release of this catecholamine. Thus, both enzymes have been associated with the pathology of dopaminergic systems and represent therapeutic targets elf enormous clinical importance. Some neuroendocrine and circadian effects of melatonin have been attributed to an antidopamimetic effect of this pineal hormone in the hypothalamus and pituitary. Furthermore, both melatonin and dopamine modulate the behavioural output of the mesencephalic dopaminergic pathways of the basal ganglia, including movement disorders. However, the biochemical basis for the tonic inhibitory effect of melatonin in the nigro-striatal pathway has been poorly delineated. Thus, this study determined whether melatonin influences dopaminergic function in the corpus striatum of the Wistar rat by modulating monoamine oxidase and catecholO- methyltransferase activity. Reciprocally, the putative existence of an intrapineal dopaminergic system was investigated by determining the effect of selective dopaminergic agents, R-( -)apomorphine, haloperidol and dopamine, on indole metabolism of the pineal gland. The akinetic state of drug-induced catalepsy was employed as an animal model of Parkinson's disease to probe the neurotransmitter systems involved in the behavioural effects of melatonin. Indole metabolism was a reliable indicator of state-dependent metabolic fluxes in pineal gland function. These included a robust diurnal and seasonal variation in N-acetylserotonin and melatonin biosynthesis, and photoperiod- and drug-induced alterations of Inftabolism. The predominant changes could be attributed to an effect on serotonin N-acetyltransferase activity and/or the melatoninl5-methoxytryptophol ratio. Pineal 5-methoxyindole biosynthesis was determined primarily by the bioavailability of the corresponding 5-hydroxyindole and its affinity for hydroxyindole-O-methyltransferase. Evidence was found for the negative feedback or paracrine control of pineal indole metabolism by melatonin. A high inter-individual variability was observed in the biosynthesis of N-acetylserotonin and melatonin biosynthesis, and the weight of the pineal glands. Accordingly, the rats could be classified as either high or low capacity producers of these two indoles. R-(-)-apomorphine and dopamine in vitro, but not acute haloperidol in vivo, had dose- and phase-dependent effects on pineal indole metabolism. The predominant effect was a suppression of the scotophase-dependent induction ofN-acetylserotonin and melatonin biosynthesis by dopamine and R-( -)-apomorphine. It is postulated that these agonists inhibited nocturnal N-acetyltransferase activity via postsynaptic pineal D2 or D2-like receptors. The observed modulatory nature of the intrapineal dopaminergic system suggests that dopamine may be involved in the long-term regulation of pineal indole biosynthesis. Several lines of evidence are presented that the activity of striatal monoamine oxidase A and catechol-O-methyltransferase, represented predominantly by the soluble isoform, is statedependent and regulated in vivo by endogenous melatonin. Firstly, both enzymes showed a daynight variation in activity. Secondly, acute and subchronic administration and photoperiod manipulation studies indicated that both exogenous and endogenous melatonin inhibited each enzyme in a chronotypic fashion, with a more robust effect against catechol- -methyltransferase. The intensity of the in vivo effects was critically dependent on the dose, duration, route and the phase-timing of administration during the light dark cycle, and the length of the exposure to constant light. Melatonin in vitro had no effect on basal or Mg2+ -induced catechol-Omethyltransferase activity. Thus, it is proposed that the in vivo effects of the hormone can be attributed to a time-dependent change in the amount of active molecules of this enzyme. In contrast, melatonin and numerous other endogenous indolic compounds were found to be reversible inhibitors of striatal monoamine oxidase A in vitro. Structure-activity modeling revealed that the 5-methoxy moiety on the indole nucleus and substitution of the free primary amine of these compounds were the principal determinants of the potency and time-dependency of inhibition. Thus melatonin most likely has a direct inhibitory effect in vivo at the level of the active site of monoamine oxidase A. Exogenous melatonin alone had no cataleptogenic potential whereas a variety of behavioural responses were observed following intraperitoneal administration of y-hydroxybutyrate. The latter responses were state-dependent with day-night variations in intensity. Furthermore, yhydroxybutyrate stimulated melatonin biosynthesis during the photophase both in vitro and in vivo. These results point to a possible involvement of melatonin in the behavioural and neurochemical effects of y-hydroxybutyrate. Thus the general conclusion is that dopamine and melatonin display functional antagonism at the level of the pineal gland and corpus striatum of the Wistar rats. Therefore melatonin may be an important homeostatic modulator of dopaminergic neurotransmission throu~out the central nervous system. Furthermore, the putative existence of a functional pineal-striatal axis would greatly strengthen the argument for a holistic concept of brain homeostasis. The ability of endogenous melatonin to regulate monoamine oxidase A and catechol-O-methyltransferase may represent an alternative strategy for the treatment of disorders associated with these enzymes.

Includes bibliographical references (leaves 166-195) Addresses the nature of the central mechanisms involved in the regulation of the circadian pattern of secretion of the pineal hormone melatonin in the highly seasonal Suffolk breed of sheep. Provides new information on the behaviour of the onset and offset of melatonin secretion under different photoperiodic conditions.

Thesis (MScMedSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Obesity has reached epidemic proportions worldwide and is currently a serious health problem. It is associated with metabolic abnormalities, oxidative stress, hypertension, insulin resistance and an increased disposition for the development of cardiovascular disease. Elucidation of the pathophysiological mechanisms underlying obesity and its relationship with metabolic and cardiovascular diseases is essential for prevention and management of these disorders. Melatonin, the pineal gland hormone, is a powerful antioxidant and has been shown to protect the myocardium against ischaemia/reperfusion (I/R) injury. Long- as well as shortterm melatonin treatment also reversed several of the harmful effects of obesity in an animal model of hyperphagia-induced obesity (DIO). However, its effects on myocardial I/R injury and intracellular signalling in obesity induced by a high fat diet (HFD) are still unknown. Aims of study: (i) To evaluate the ability of a high fat diet (HFD) to induce obesity in rats. Apart from evaluating its effects on the biometric parameters and resistance to ischaemia/reperfusion injury (as indicated by infarct size in regional ischaemia and functional recovery after global ischaemia), special attention will be given on the interplay between adiponectin, AMPK, leptin, and FFA in this model. (ii) To evaluate the effect of daily oral administration of melatonin to rats on the HFD as well as their littermate controls, on the parameters listed above as well as on the development of obesity. In this study melatonin will be administered from the onset of the feeding of the high fat diet. Methods: Male Wistar rats were divided into 4 groups: (i) control rats (receiving normal rat chow) (C); (ii) control rats receiving melatonin (CM); (iii) obese rats (receiving HFD) (HFD); (iv) obese rats receiving melatonin (HM). Animals were kept on the diet for 16 weeks and melatonin treatment (10mg/kg/day, added to the drinking water) started at the onset of the feeding. Following feeding and treatment, the animals were grouped into fasted/ non-fasted of which biometric parameters were recorded and blood collected at the time of sacrifice for metabolic and biochemical assays. Hearts were perfused in the working mode for evaluation of myocardial function and infarct size determination after exposure to 35min regional ischaemia/60min reperfusion. For study of intracellular signaling, hearts were perfused in the working mode, subjected to 20min global ischaemia/10min reperfusion and freeze-clamped for Western blotting. Plasma leptin, adiponectin, free fatty acid, triglycerides, total cholesterol, phospholipids, conjugated dienes and thiobarbituric reactive substances (TBARS) levels were determined. Several kinases were investigated including, the RISK (reperfusion injury salvage kinase) (PKB/Akt and ERK p44/42) and SAFE (survivor activating factor enhancement) (STAT-3) pathways, AMPK and JNK under baseline conditions or following 10 min reperfusion. In addition, expression of UCP-3 and PGC1-α was determined. Results: Significant increases in body weight, visceral fat, blood glucose, insulin, HOMA index and leptin and a reduction in adiponectin levels were observed in the fasted high fat diet (HFD) group when compared with controls (C). Significant increases in free fatty acid and triglyceride levels were also noted the HFD group while other serum lipid parameters, including TBARS, remained unchanged. No differences in functional recovery during reperfusion or infarct size after exposure to 35 min regional ischaemia, as well as functional recovery during reperfusion after 20 min global ischaemia were observed between the control and HFD groups. Baseline and 10 min reperfusion data were similar for the RISK and SAFE pathway kinases for the control vs HFD groups. The HFD also had no effect on the expression and phosphorylation of myocardial AMPK and JNK, as well as on the expression of UCP-3 and PGC1-α, when compared to the controls. Treatment with melatonin significantly reduced body weight, visceral fat, blood glucose, HOMA index and serum leptin levels in HFD treated groups, while having no effect on the lipid profile. Although melatonin significantly reduced infarct size in both control [% of area at risk: 20.59 ± 2.29 (CM) vs 38.08 ± 2.77 (C)] and high-fat diet groups [% of area at risk: 11.43 ± 2.94 (HM) vs 38.06 ± 3.59 (H)], it was without effect on myocardial functional recovery during reperfusion. Melatonin had no effect on the intracellular signaling pathways studied. Conclusions: The HFD proved to be a useful model of diet-induced obesity with a more pronounced impact on biometric and metabolic changes compared to the DIO model. Long-term melatonin treatment successfully prevented the development of metabolic abnormalities associated with the high fat diet and obesity as well as significantly reduced myocardial infarct size. The mechanisms involved in melatonin-induced cardioprotection in obesity have not been fully elucidated in this study and require further investigation. However, the anti-obesogenic and cardioprotective properties of melatonin were very significant indeed and support the suggestion of this hormone as a potential tool in the treatment of obesity and associated cardiovascular complications.
AFRIKAANSE OPSOMMING: Inleiding: Vetsug (obesiteit) het wêreldwyd epidemiese afmetings aangeneem en word tans as ‘n ‘n ernstige gesondheidsprobleem beskou. Vetsug word geassosieer met metaboliese afwykings, oksidatiewe stres, hipertensie, insulienweerstandigheid en is‘n belangrike risikofaktor vir die ontwikkeling van kardiovaskulêre siekte. Ten spyte hiervan, het onlangse studies ‘n gunstige effek van vetsug op die uitkomste van miokardiale infarksie in pasiënte gerapporteer, die sg obesiteitsparadoks. Kennis van die patofisiologiese meganismes onderliggend aan vetsug en die ontstaan van metaboliese afwykinge en hartsiekte is noodsaaklik vir die voorkoming en behandeling van hierdie toestande. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige antioksidant en vry radikaal opruimer. Dit is voorheen aangetoon dat dit die hart teen iskemie/herperfusie (I/H) besering kan beskerm en sommige van die skadelike gevolge van vetsug in diermodelle kan omkeer. Die effek van melatonien op miokardiale I/H besering en intrasellulêre seintransduksie prosesse in vetsug geïduseer deur ‘n hoë vet dieet is egter nog onbekend. Doelstellings: (i) Die ontwikkeling en karakterisering van ‘n nuwe model van vetsug en insulienweerstandigheid geïnduseer deur 'n hoë vet dieet (HVD) en die evaluering van die effek daarvan op miokardiale I/H besering en die gepaardgaande intrasellulêre seintransduksieprosesse; (ii) Bepaling van die effek van daaglikse toediening van melatonien aan rotte op die HVD sowel as aan kontroles op ‘n standard dieet, op die ontwikkeling van dieet-geïnduseerde metaboliese veranderinge, miokardiale infarktgrootte en funksionele herstel na koronêre arterie afbinding, sowel as intrasellulêre seintransduksie. Metodiek: Vier groepe van manlike Wistar rotte is bestudeer: (i) kontrole rotte (op‘n standaard dieet) (K); (ii) kontrole rotte op ‘n standard dieet plus melatonien (KM); (iii) dieetrotte (op‘n HVD); (iv) HVD rotte wat melatonien ontvang (HM). Die HVD en melatonien (10mg/kg/dag in die drinkwater) is vir 16 weke toegedien. Na die periode van behandeling, is die diere in vastende en nie-vastende groepe verdeel, die biometriese parameters genoteer en bloedmonsters vir metaboliese en biochemiese bepalings versamel, tydens verwydering van die harte. Harte is geperfuseer volgens die werkhartmodel vir bepaling van miokardiale funksie en infarktgrootte na blootstelling aan 35min streeksiskemie. Vir evaluering van intrasellulêre seintransduksie, is geperfuseerde werkende rotharte blootgestel aan 15min globale iskemie/10 min herperfusie en gevriesklamp vir latere analises volgens die Western kladtegniek.hart. Serum leptien, adiponektien, vryvetsure, trigliseried, totale cholesterol, fosfolipiede, gekonjugeerde diene en tiobarbituursuur reaktiewe stowwe (TBARS) is bepaal. Met gebruik van Western kladtegniek, is die aktivering en/of uitdrukking van die RISK (PKB/ Akt en ERK p44/42) en SAFE (STAT-3) seintransduksiepaaie, AMPK, JNK, UCP-3 en PGC1-α, onder basislyn toestande of na 10 min herperfusie bestudeer. Resultate:‘n Beduidende toename in liggaamsgewig, visserale vet, die HOMA indeks, insulien en leptien vlakke is in die HVD groep waargeneem vergeleke met die kontrole (K) rotte. Adiponektien vlakke was laer in die HVD groep. Die HVD groep is ook gekenmerk deur ‘n beduidende styging in serum vryvetsuur en trigliseried vlakke, terwyl die ander lipied parameters, insluitende die TBARS vlakke, onveranderd was. Infarktgrootte en funksionele herstel tydens herperfusie na blootstelling aan 35 min streeksiskemie, asook funksionele herstel tydens herperfusie na 20 min globale iskemie het nie verskil tussen harte van die kontrole en HVD rotte nie. Aktivering van PKB/Akt, ERK p44/p42, STAT3, AMPK en JNK by basislyn en na 10 min herperfusie was soortgelyk in die kontrole en HFD groepe. Die HVD het ook geen effek op die uitdrukking van UCP-3 en PGC1-α in vergelyking met die kontrole gehad nie. Behandeling met melatonien het die liggaamsgewig, visserale vet, bloedglukose, HOMA indeks en serum leptien vlakke in die HVD groepe statisties beduidend verlaag, terwyl dit geen invloed op die lipiedprofiel gehad het nie. Melatonien behandeling het die miokardiale infarktgrootte beduidend en tot dieselfde mate verminder in beide kontrole [20.59 ± 2.29 (KM) vs 38.08 ± 2.77% (K)] en HVD groepe [11.43 ± 2.94 (HM) vs 38.06 ± 3.59% (HVD)]. Geen verskille is egter tussen die funksionele herstel gedurende herperfusie van die behandelde en onbehandelde kontrole en HVD groepe waargeneem nie. Melatonien het ook geen uitwerking op die intrasellulêre seintransduksiepaaie gehad nie. Gevolgtrekkings: Die resultate het getoon dat die HFD 'n goeie model van dieetgeïnduseerde vetsug en insulien weerstandigheid ontlok, met 'n meer uitgesproke impak op biometriese en metaboliese veranderinge as die voorheen gebruikte hoë-sukrose dieet. Langtermyn melatonien- behandeling het die ontwikkeling van metaboliese abnormaliteite geassosieer met die HVD, voorkom, asook miokardiale infarktgrootte na koronêre afbinding beduidend verminder. Die meganismes betrokke in melatonien-geïnduseerde miokardiale beskerming moet egter in meer detail ondersoek word. Die resultate verkry steun die voorstel dat melatonientoediening voordelig sal wees in die behandeling van vetsug en sy kardiovaskulêre komplikasies.

RESUME GENERAL

Contexte :une association entre exposition prolongée aux champs magnétiques (CM) d’extrêmement basses fréquences (ELF) et risque sanitaire a été établie pour la leucémie infantile (CM 50/60 Hz de l’électricité, RR = 2,0 pour ≥ 0,4 µT d‘intensité moyennée dans le temps) et est suggérée pour le décès par maladie d’Alzheimer (CM 50/60 Hz, CM 16,7 Hz des voies ferrées pour 21 µT d’intensité moyennée dans le temps) et pour certaines hémopathies chez l’adulte (CM 16,7 Hz). Ces associations restent inexpliquées à ce jour. Sur base d’observations animales (effets des CM ELF sur la sécrétion de mélatonine) d’une part, et de la sensibilité magnétique confirmée des cryptochromes (régulateurs des biorythmes) d’autre part, il a été suggéré que ces associations puissent être dues à une perturbation des biorythmes par les CM ELF. Selon les instances internationales, une intensité > 1 mT est requise pour l’existence d’effets biologiques.

Objectifs et méthode :sur base d’une revue exhaustive de la littérature et de modèles théoriques reconnus, le présent travail développe certains mécanismes possibles pour un effet perturbateur des biorythmes par les CM ELF. L’impact en santé publique de cette hypothèse est ensuite évalué. Enfin, des protocoles sont proposés pour sa mise à l’épreuve, tenant compte des mécanismes envisagés.

Résultats :la possibilité existe d’une interaction des oscillations ELF de l’intensité et/ou de l’orientation du CM (somme vectorielle du CM ELF et du CM terrestre ou CMT) avec les cryptochromes rétiniens. Chez l’animal magnétosensible (dont le rongeur), une perturbation des biorythmes pourrait être consécutive à un mécanisme non spécifique de perturbation sensorielle. Toute observation animale pourrait donc ne pas être extrapolable à l’Homme. Chez ce dernier, une perturbation des biorythmes pourrait être causée par les oscillations de l’intensité du CM (peut- être dès < 100 µT d’intensité de CM ELF). Une telle perturbation pourrait aussi être causée par les variations spatiales de l’intensité du CMT qui existent dans l’environnement résidentiel (proximité de structures métalliques). Par ailleurs, dans l’éventualité de l’existence, chez l’Homme également, d’une sensibilité directionnelle basée sur les cryptochromes rétiniens, les oscillations de l’orientation du CM pourraient alors aussi interférer avec ces cryptochromes (peut-être dès ≤ 10 µT). Dans l’hypothèse où une telle interférence affecte les biorythmes, seules pourraient alors être concernées les oscillations dont l’amplitude atteint plusieurs degrés d’angle. Un tel mécanisme ne pourrait donc s’appliquer à la relation entre CM ELF et leucémie infantile que dans l’éventualité où les intensités les plus élevées (+ 1 à 2 SD) de CM ELF y jouent un rôle. Au cas où l’hypothèse de la perturbation des biorythmes par les CM ELF se voyait confirmée, d’autres troubles de santé seraient alors concernés et d’autres sources de CM seraient en cause, tels les CM statiques d’intensité variable émis par les lignes de transport électrifié. Les paramètres d’exposition considérés devraient inclure l’orientation relative CM ELF/CMT, mais aussi l’intensité locale du CMT (facteur à la fois déterminant et confondant dans la présente hypothèse). L’expérimentation animale devrait investiguer l’expression des clock genes. L’expérimentation humaine devrait investiguer les biorythmes chez l’enfant. Et l’épidémiologie devrait investiguer l’incidence de troubles liés à une perturbation des biorythmes en relation avec l’exposition aux CM ELF ainsi qu’aux variations locales de l’intensité du CMT.

Conclusions :malgré les incertitudes persistantes quant aux fonctions précises des cryptochromes de la rétine humaine et quant à l’exactitude des modèles théoriques qui décrivent les interactions entre CM et cryptochromes, certains mécanismes paraissent possibles pour une interaction entre CM ELF et biorythmes. En l’absence persistante d’alternative valide pour l’explication de l’association entre CM ELF et leucémie infantile, l’hypothèse de la perturbation des biorythmes par ces CM paraît devoir être investiguée plus avant, mais en tenant compte des variations locales d’intensité du CMT.

Background: An association between prolonged exposure to extremely low frequency (ELF) magnetic fields (MF) and health risk has been established for childhood leukemia (50/60 Hz MF of electricity, RR = 2.0 for ≥ 0.4 µT of time-averaged intensity) and is suggested for death by Alzheimer's disease (50/60 Hz MF, 16.7 Hz MF of railways at 21 µT of time-averaged intensity) and for some hematologic malignancies in adults (16.7 Hz MF). These associations remain unexplained so far. Based on animal studies (effects of ELF MF on melatonin secretion) on the one hand, and on the confirmed magnetic sensitivity of cryptochromes (regulators of biorhythms) on the other hand, it has been suggested that these associations may be due to a disruption of biorhythms by ELF MF. From current data, however, biological effects seem only possible at > 1 mT of intensity.

Objectives and methods: on the basis of an exhaustive literature review and with use of recognized theoretical models, this paper develops some possible mechanisms for disruption of biorhythms by ELF MF. The public health impact of this hypothesis is then evaluated. Finally, protocols are proposed for the testing of it, with taking into account the proposed mechanisms.

Results: an interaction seems possible between ELF oscillations of the intensity and/or the orientation of the ambient MF (the vector sum of both the ELF MF and the geomagnetic field or GMF) with retinal cryptochromes. In magnetosensitive animals (including rodents), disruption of biorhythms may then be secondary to a non-specific mechanism of sensory disturbance. All animal observation could therefore not be extrapolated to humans. In the latter, on his turn, a disruption of biorhythms may be caused by the oscillations of the MF intensity (perhaps from <100 µT of ELF MF intensity). Such disruption could also be caused by spatial variations of the intensity of the GMF that exist in residential environment (near steel structures). Moreover, in case of the existence in humans (like in animals) of a directional sensitivity based on retinal cryptochromes, then the oscillations of the MF orientation also could interfere with these cryptochromes (perhaps from ≤ 10 µT). In the event that such interference affects biorhythms, only oscillations of several degrees of amplitude would then be concerned. As a consequence, such a mechanism could apply to the relation between ELF MF and childhood leukemia only in the event that the highest MF intensities (Mean + 1-2 SD) also play a role in that relation. In the event the hypothesis of disruption of biorhythms by ELF MF is confirmed, other health problems would be concerned and other kind of MF would be involved, such as the static MF of variable intensity that are emitted by the lines of electrified transport. The considered exposure parameters should include the relative orientation of ELF MF and GMF, but also the local intensity of GMF (both determining factor and confounder in this case). Animal experiments should investigate the expression of clock genes. Human experimentation should investigate biorhythms in children. And epidemiology should investigate the incidence of disorders related to disruption of biorhythms in relation to exposure to ELF MF as well as to local variations in the intensity of the GMF.

Conclusions: Despite the persisting uncertainties about the precise functions of retinal cryptochrome as well as about the accuracy of the theoretical models that describe the interactions between MF and cryptochromes, some mechanisms seem possible for an interaction between ELF MF and biorhythms. In the persisting absence of valid alternative explanation for the association between childhood leukemia and ELF MF, the hypothesis of biorhythm disturbance by ELF MF deserves further investigation, however with taking into account local intensity variations of the GMF.

Doctorat en Sciences de la santé publique
info:eu-repo/semantics/nonPublished

Ph.D., Faculty of Science, University of the Witwatersrand, 2011
This study investigated the effect of exogenous melatonin administration on transient global cerebral ischemia and adult neurogenesis in adult male Sprague- Dawley rats. It also determined serum melatonin concentrations in all the experimental groups and established any effect of melatonin on estimated total granule cell numbers. Adult male Sprague-Dawley rats were divided into eight groups with each group consisting of 6 rats (n = 6). Post-induction time durations of 72 hours and 7 days was used. Single dose of 5 mg/kg exogenous melatonin was administered at each phases of 30 minutes before and after a 10 minutes transient bilateral occlusion of the common carotid arteries in the different groups, followed by reperfusion. Rats were anesthetized with 20 mg/kg of ketamine and 2.5 mls of blood was collected via cardiac puncture for estimation of serum melatonin concentration using commercially prepared radioimmunoassay ELISA kit. Free floating brain sections cut at 50 μm were immunostained for Ki-67, marker for proliferating cells. The total granule cell number in the dentate gyrus was estimated using the optical fractionator method on plastic embedded brain sections. Mean melatonin concentration (pg/mol) was 268.54 ± 28.73 (72 hours) and 277.83 ± 28.73 (7 days) compared to the sham control; 266.94 ± 37.6 and non surgical 262.96 ± 23.85 respectively. Differences in the concentration were not statistically significant (P<0.05). Histological finding indicated neuropil disruption with potentiation of restoration as the post ischemia days progressed in the melatonin administered groups. The estimated total granule cell number in the dentate gyrus of the hippocampus was not affected by exogenous melatonin administration. However, there was potentiation in proliferations of the neurogenic niche in the dentate gyrus of the hippocampus demonstrating a very strong indications that melatonin enhanced the generations of proliferating cells in adult male Sprague-Dawley rats.

Bibliography: leaves 162-197.
vii, 216 leaves : ill. ; 30 cm.
To determine the effects of melatonin on sleepiness and body temperature under conditions that better approximated the endogenous melatonin profile.
Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1998?

Includes bibliographical references (leaves 166-195)
xxi, 195 leaves : ill. ; 30 cm.
Addresses the nature of the central mechanisms involved in the regulation of the circadian pattern of secretion of the pineal hormone melatonin in the highly seasonal Suffolk breed of sheep. Provides new information on the behaviour of the onset and offset of melatonin secretion under different photoperiodic conditions.
Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1989

Graduation date: 2006
Most vertebrates exhibit seasonality in many life history traits. Such seasonal rhythms are temporally organized via the transduction of environmental cues (e.g., photoperiod, temperature) into appropriate endocrine signals. However, among ectothermic vertebrates that undergo continuous winter dormancy, temperature is the only environmental cue available for synchronizing seasonal rhythms. Most intriguing is that in species where reproduction occurs immediately following spring emergence, the associated changes in neurophysiology and behavior that accompany reproduction likely occur during winter dormancy. The purpose of this dissertation research was to explore the mechanisms by which temperature cues affect the chronobiology and seasonal reproduction of red-sided garter snakes (Thamnophis sirtalis parietalis). Because of their roles in circadian organization and energy balance, melatonin and corticosterone are likely hormonal components of these time-keeping systems. I first characterized the interactions between melatonin and corticosterone to better understand the hormonal mechanisms facilitating temperature-induced reproduction. Melatonin and corticosterone additively inhibit reproductive behavior during the spring mating season. Experimental manipulations with a serotonin receptor antagonist suggest the mechanism underlying these effects involves a serotonin-regulated system. Although melatonin does not influence corticosterone responses to capture stress, capture stress significantly increases melatonin concentrations. To investigate the functional significance of these interactions in regulating temperature-induced reproduction, I measured body temperatures of snakes as well as circadian melatonin and corticosterone cycles during winter dormancy and spring emergence using a combination of field and laboratory experiments. Surprisingly, an increase in body temperature is not necessary for emergence from winter dormancy. Rather, critically low temperatures may serve as a zeitgeber entraining an endogenous circannual cycle that regulates emergence. Decreased environmental temperatures, in the absence of changing photoperiod cues, modulate circadian melatonin and corticosterone rhythms during hibernation. Such temperature-induced changes in hormone rhythms may facilitate seasonal reproductive behavior following spring emergence. Furthermore, a phase-shift in corticosterone rhythms during the mating season may regulate the seasonal transition between reproductive and non-reproductive states in red-sided garter snakes. Such studies investigating the environmental and hormonal mechanisms underlying time-keeping systems may provide valuable insight into the potential impact of environmental perturbations (e.g., climate change) on seasonal rhythms in physiology and behavior.

L’objectif de ce projet de recherche était de vérifier la présence de changements de sensibilité de la rétine et du système circadien suite à deux semaines d'exposition à un milieu faiblement ou fortement éclairé, dans des conditions contrôlées en laboratoire. De plus, comme un changement de sensibilité peut modifier l'ajustement du système circadien au cycle jour-nuit extérieur, nous voulions également vérifier si la phase circadienne serait modifiée par le traitement et si la vigilance et l’humeur seraient affectées. Dix sujets ont été exposés à de la lumière tamisée (70 lux [LT]) et 10 ont été exposés à de la lumière vive (3000 lux [LV]) pendant 12 jours consécutifs en laboratoire de 8h45 à 19h00 tous les jours. L’exposition à la lumière a été mesurée 5 jours avant l’entrée au laboratoire dans l’habitat naturel du sujet et pendant la période en laboratoire à l’aide de l’Actiwatch-L®. La sensibilité rétinienne a été mesurée avant et après le traitement lumineux, par un électrorétinogramme (ERG) et la sensibilité circadienne, par le test de suppression de mélatonine salivaire. Tout au long du protocole, la vigilance, la somnolence et l'humeur ont été évaluées à plusieurs moments de la journée à intervalles prédéterminés. Après 12 jours d’exposition en lumière contrôlée, l’amplitude de l’onde-a au Vmax à l’ERG photopique a diminué en LV alors qu’elle a augmenté en LT. À l’ERG scotopique, une différence de sensibilité rétinienne (log K) entre les groupes avant le traitement expérimental s’est amenuisée à la fin du traitement (p=.053). La suppression de mélatonine après 90 minutes d’exposition au test de suppression a diminué en LV alors qu’il n’y a pas eu de modification en LT, cependant cette interaction n’était pas significative (p=.16). La phase circadienne des sujets exposés à LV a été devancée de 58 minutes (p=.04) alors qu’elle a été retardée de 26 minutes en LT (p=.32). Les mesures de vigilance subjective (EVA) ont indiqué que les sujets LV se considéraient plus éveillés que les sujets LT après le traitement (p=.02). Par contre, aucune différence n’est apparue quant aux mesures de performance psychomotrice ni de l’humeur. L’histoire lumineuse n’a pas modifié la sensibilité rétinienne dans le sens prévu par les hypothèses alors qu’il y a eu une tendance vers une augmentation de la sensibilité circadienne en condition de lumière tamisée. L’amélioration de la vigilance subjective après l’exposition en LV n’a pas été soutenue par les résultats de la performance psychomotrice. L’histoire lumineuse n’a eu aucun effet sur l’humeur des sujets. Cette étude souligne l’importance d’utiliser des mesures permettant de départager les effets immédiats d’un traitement lumineux des effets à long terme autant sur le plan rétinien que circadien. Il reste également complexe d’étudier en laboratoire des changements adaptatifs qui se produisent dans le milieu naturel en raison du confinement et des modifications physiologiques et psychologiques pouvant y être associées.
The purpose of this study was to evaluate the impact of two weeks exposure in a dim or bright light environment on retinal and circadian sensitivity to light in a controlled laboratory setting. Given that a change in sensitivity to light could modify the circadian adjustment to the external light-dark cycle, it was expected that the circadian phase would be modified with the light treatment and have an effect on alertness and mood. Ten participants were exposed to a dim light (DL) environment (70 lux) and 10 participants to a bright light (BL) environment (3000 lux) 10 hours per day for 12 consecutive days. Light exposure was measured 5 days prior to the onset of the experiment in the subject’s natural environment and during the entire laboratory experiment with an Actiwatch-L®. Retinal function was assessed with the electroretinogram (ERG). Circadian light sensitivity was evaluated with a salivary melatonin suppression test. Retinal and circadian sensitivity measures were taken before and after the experimental condition. Alertness, sleepiness and mood were measured several times per day at fixed intervals. After 12 days of controlled light exposure, the amplitude of amax of the photopic ERG was decreased in BL whereas it was increased in DL. In scotopic ERG, there was a difference in the retinal sensitivity (log K) between the two groups before light treatment that disappeared at the end of light exposure (p=.053). The percentage of melatonin suppression after 90 minutes exposure to the melatonin suppression test was decreased in BL while it did not changed in DL condition. This interaction, however, did not reach significance (p=.16). We measured a 58 minutes phase advance in the BL condition (p=.04) and a 26 minutes phase delay in DL (p=.32). Measures of subjective vigilance (EVA) suggested that BL subjects were more alert after the light treatment than DL subject (p=.02). However, there was no difference in the psychomotor vigilance task or mood. Light history did not modify the retinal sensitivity as predicted by the hypotheses. However, there was a trend toward an increased circadian sensitivity in the dim light condition. The improvement of subjective vigilance in the BL condition was not supported by the results at the psychomotor vigilance task. Light history had no effect on the mood of the subjects. Long-term effects of a light treatment are difficult to isolate from shorter direct effects of light. Moreover, the study of adaptative environmental changes that spontaneously appeared in the field are possibly masked in a laboratory setting where confinement could induce physiological and psychological changes.