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Violence against women : impact on reproductive health and pregnancy outcomeSchoeman, Jeanne 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Introduction
Worldwide, up to 25% of women are assaulted during pregnancy, with estimates
varying between populations. Violence has been associated with adverse
pregnancy outcome, including preterm birth, abruptio placentae and low birth
weight. Among the Coloured population of the Western Cape the incidence of
spontaneous preterm birth is 20%, compared to the global figure of 10%.
Overall, the rate of preterm labour has not dropped over the past 40 years and
no clearer answer as to a specific cause has been found.
The objective of this study was to determine whether patients who deliver
preterm experience more domestic violence than those who deliver at term.
Methods
Two groups of patients were assessed. Firstly, patients who spontaneously
delivered between 24 and 33 weeks (24wOd - 33w6d), who were admitted for
suppression of active labour after 24 weeks, or who experienced placental
abruption before 34 weeks, were screened for domestic violence using the
"Abuse Assessment Screen". A second group of women, attending a local
Midwife Obstetric Unit with uncomplicated pregnancies, completed the same
questionnaire. The questionnaires were all administered by the same person
(J.S.) after written informed consent was given.
Results
A total of 229 patients were interviewed, 99 in the low risk (LR) and 130 in the
preterm labour (PTL) group, which included 23 women with abruptio placentae.
The PTL group experienced significantly more violence throughout their lives
than the LR group (59.7% vs. 40.4%, p = 0.038). Experiences of violence within the last year or during the pregnancy did not reach statistical significance
between the two groups, although the numbers were higher for the PTL group.
The PTL group smoked significantly more cigarettes per day (p = 0.009), used
more alcohol (p < 0.001) and had a higher incidence of syphilis than the LR
group (p = 0.005). These differences remained the same when the abruptio's
were analyzed as a separate group.
Conclusions: Women who delivered preterm did experience more violence at
some point in their lives and were also more likely to engage in high-risk
behaviour. Violence alone does not seem to cause PTL directly, but is part of a
low socioeconomic lifestyle. The fact that the alcohol use is so high among these
women is a problem that needs to be addressed, but once again, it is possibly
the result of deeper social problems. The need for education on values and
respect, family planning use and low risk sexual behaviour is once again
challenged. / AFRIKAANSE OPSOMMING: GEWELD TEEN VROUE -IMPAK OP REPRODUKTIEWE GESONDHEID EN
UITKOMS VAN SWANGERSKAP
Inleiding
Daar word beraam dat tot 25% van alle swanger vroue aangerand word, maar
die insidensie wissel tussen verskillende populasies. Ervarings van geweld kan
'n direkte of indirekte oorsaak wees van swak verloskundige uitkoms wat
voortydse kraam, abruptio placentae en lae geboortegewig insluit. In die Wes-
Kaap, onder die Kleurlingbevolking, is die insidensie van voortydse kraam 20%,
wat swak vergelyk met die wêreldwye insidensie van 10%. Gedurende die laaste
40 jaar het die voorkoms van voortydse kraam nie verminder nie en geen
deurbrake is gemaak t.o.v die oorsaak van die probleem nie. Die doel van
hierdie studie was om te bepaal of vroue wat prematuur verlos moontlik meer
geweld ervaar as vroue wat op normale swangerskapsduur verlos.
Metodes
Twee groepe vroue is bestudeer. Die eerste groep het vroue ingesluit wat
spontaan verlos het tussen 24 en 33 weke (24wOd - 33w6d) of vroue wat na 24
weke swangerskapsduur toegelaat is vir onderdrukking van kraam. Vroue met
plasentale loslating (abruptio placentae) voor 34 weke, sonder onderliggende
hipertensiewe toestande, was ook ingesluit in die groep. Daar is m.b.v. 'n
vraelys ("Abuse Assessment Screen") bepaal watter van die vroue gesinsgeweld
ervaar het. Die tweede groep het vroue ingesluit met ongekompliseerde
swangerskappe en wat by 'n nabygeleë kliniek voorgeboortesorg ontvang het.
Hulle is ook gevra om die vraelys te voltooi en is opgevolg om die uitkoms van
hulle swangerskappe te noteer. Die vraelyste is almal deur een persoon (J.S.)
aan die vroue voorgelê nadat hulle ingeligte, skriftelike toestemming gegee het. Resultate
'n Totaal van 229 vroue was ingesluit, 99 in die lae risiko (LR) groep en 130 in
die voortydse kraam (VK) groep, waarvan 23 abruptio placentae gehad het. In
vergelyking met die LR groep, het die VK groep het betekenisvol meer geweld in
hulle leeftyd ervaar (59.7% teenoor 40.4%, p = 0.038). Geweld wat tydens die
afgelope jaar of tydens die swangerskap ervaar is, het nie betekenisvol verskil
tussen die twee groepe nie, alhoewel die getalle hoër was vir die VK groep. Die
VK groep het betekenisvol meer sigarette per dag gerook (p = 0.009), meer
alkohol gebruik (p < 0.001) en het 'n hoër insidensie van sifilis gehad as die LR
groep (p = 0.005). Hierdie verskille was steeds beduidend nadat dié met
abruptio placentae as 'n aparte groep geanaliseer is.
Gevolgtrekking
Die vroue wat prematuur verlos het, het meer emosionele en fisiese geweld in
hulle leeftyd ervaar en is meer geneig om 'n ongesonde leefstyl te handhaaf.
Geweld blyk nie 'n direkte oorsaak van voortydse kraam te wees nie, maar gaan
gepaard met 'n lae sosio-ekonomiese lewensstyl. Die hoë insidensie van
alkoholgebruik onder swanger vroue is 'n probleem wat aangespreek moet word,
maar dit is waarskynlik die manifestasie van dieper emosionele probleme.
Opvoeding in terme van waardes en respek, gesinsbeplanning en veilige
seksuele gedrag is gevolglik 'n noodsaaklikheid.
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Gender selection: separation techniques for X- and Y-chromosome bearing human spermatozoaVan Der Linde, Michelle 12 1900 (has links)
Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Preconceptual sex selection is an ethically justifiable process whereby X- and Y-chromosome bearing spermatozoa are isolated prior to fertilization of the oocyte in order to generate either a male or a female offspring. Although various separation techniques are available, none can guarantee 100% accuracy. There are various physiological differences between X- and Y-chromosome bearing spermatozoa which can be used to separate these two populations of sperm.
For the purpose of this study, X- and Y-chromosome bearing spermatozoa were separated based on (1) their respective abilities to remain viable when subjected to adverse environments, including extreme pH values, increased temperatures and various hydrogen peroxide (H2O2) concentrations; (2) the ability of Y-chromosome bearing spermatozoa to swim faster and/or more progressively than X-chromosome bearing spermatozoa; and (3) the X-chromosome bearing spermatozoa’s increased size and weight when compared to the Y-chromosome bearing spermatozoa.
The efficacy of live and dead cell separation through (i) Magnetic Antibody Cell Separation (MACS) and (ii) a modified swim-up technique was also assessed and compared. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. Sperm motility (CASA) and viability (eosin/nigrosin) was assessed before and after each intervention. Ethical clearance for this study was granted by the Health Research Ethics Committee 1 (Ethics #: S13/04/068).
The results indicated successful enrichment of X-chromosome bearing spermatozoa upon incubation in acidic media, increased temperatures, and H2O2. In contrast, Y-chromosome bearing spermatozoa were successfully enriched through a direct swim-up method as well as discontinuous gradient centrifugation.
In conclusion, this study demonstrated the potential role for physiological differences between X- and Y-chromosome bearing spermatozoa in the development of preconceptual gender selection through sperm sorting. / AFRIKAANSE OPSOMMING: Prekonsepsie geslagselektering is 'n eties regverdigbare proses waardeur X- en Y- chromosoom draende spermatosoë geïsoleer word voordat bevrugting van die oösiet plaasvind, om óf 'n manlike óf 'n vroulike nageslag te genereer. Alhoewel verskeie skeidingstegnieke beskikbaar is, kan geeneen 100% akkuraatheid waarborg nie. Daar bestaan verskeie fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë wat skeiding van hierdie twee groepe spermatosoë moontlik kan maak.
Vir die doel van hierdie studie is skeidingsmetodes vir die X- en Y- chromosoom draede spermatosoë gebaseer op (1) hul onderskeie vermoëns om lewensvatbaar te bly tydens blootstelling aan ‘n ongunstige milieu, insluitend ekstreme pH waardes, verhoogde temperature en verskeie waterstofperoksied (H2O2) konsentrasies; (2) die vermoë van die Y-chromosoom draende spermatosoon om vinniger en/of meer progressief as X-chromosoom draende spermatosoë te swem; en (3 ) die X-chromosoom draende spermatosoon se verhoogde grootte en gewig in vergelyking met die Y- chromosoom draende spermatosoon.
Die effektiwiteit van die (i) Magnetiese Anti-liggaam Sel Skeidingstegniek (MACS) en (ii) 'n aangepaste weergawe van die op-swem tegniek om lewendige en dooie selle te skei is ook bepaal en vergelyk. Veranderinge in die geslagschromosoom verhouding van die monsters is bepaal deur dubbel-etiket fluoresensie in situ hibridisering (FISH) voor en na verwerking. Spermmotiliteit (CASA) en lewensvatbaarheid (eosien/nigrosin) is bepaal voor en na elke intervensie. Etiese goedkeuring vir hierdie studie is verleen deur die Gesondheids-Navorsingsetiekkomitee 1 (Etiese # : S13/04/068).
Die resultate dui suksesvolle verryking van X-chromosoom draende spermatosoë deur inkubasie in suur media, verhoogde temperature, en H2O2. Y-chromosoom draende spermatosoë is verryk deur middel van 'n direkte op-swem metode sowel as diskontinue gradiënt sentrifugering .
Ten slotte, hierdie studie toon die potensiële rol vir fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë in die ontwikkeling van prekonsepsie geslagselektering metodes deur skeiding van X- en Y-chromosoom draende sperme.
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Effects of insulin and leptin on human spermatozoa function and their cross-talk with nitric oxide and cytokinesLampiao, Fanuel 12 1900 (has links)
Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In recent years there has been an increase in obesity and diabetes mellitus (DM).
These conditions have for a long time been associated with infertility. Obesity is
characterized by high levels of circulating leptin and cytokines as well as insulin
resistance. Type I DM is associated with low or no insulin whereas, Type II DM is
characterised by insulin resistance. As the prevalence of obesity and DM continues
to rise, it is likely that the incidence of infertility associated with these pathological
conditions will likewise increase. The effects of insulin and leptin on male
reproductive function have been reported on the endocrine and spermatogenesis
level, but their effects on cellular level of human ejaculated spermatozoa are yet to
be elucidated.
This study presents data on the role of insulin and leptin on human ejaculated
spermatozoa and their interaction with cytokines and nitric oxide. In the first part of
the study, we established the suitable concentrations of glucose, insulin and leptin
that could be administered to human spermatozoa in vitro. Glucose concentration of
5.6 mM was chosen as the suitable concentration to be administered to human
spermatozoa because it has previously been reported in the literature; furthermore, it
is within the range of the physiological glucose levels found in the blood of fasting
humans. Insulin and leptin concentrations of 10 μIU and 10 nmol were chosen
respectively because they gave much improved sperm function and this was within
the range of insulin and leptin levels previously measured in human ejaculated
spermatozoa. This was followed by investigating the signalling pathway of insulin and
its beneficial effects on human spermatozoa function. Endogenous insulin secretion
from human ejaculated spermatozoa was blocked by nifedipine and its receptor
tyrosine phosphorylation effects were inhibited by erbstatin while phosphatidylinositol
3-kinase (PI3K) phosphorylation activity was inhibited by wortmannin. Exogenous
insulin administration significantly increased human sperm motility parameters as
well as the sperm ability to acrosome react. The inhibition of endogenous insulin
release from spermatozoa as well as the inhibition of the insulin receptor substrate
(IRS) tyrosine phosphorylation significantly decreased motility parameters and the
ability of spermatozoa to acrosome react.
The study also investigated the effects of insulin and leptin on human sperm motility,
viability, acrosome reaction and nitric oxide (NO) production. Both insulin and leptin
significantly increased sperm motility parameters, acrosome reaction and NO
production. The NO production induced by insulin and leptin was via PI3K signalling
as evidenced by a reduction in NO levels when PI3K activity was inhibited by
wortmannin. To investigate whether insulin and leptin could improve motility
parameters of asthernozoospermic and teratozoospermic spermatozoa, the
spermatozoa were separated into two fractions by means of a double density
gradient technique. The gradient system was able to separate spermatozoa into high
morphologically abnormal and less motile spermatozoa similar to that of
asthernozoospermic and teratozoospermic patients as well as a more motile fraction.
Insulin and leptin significantly increased the motility parameters of spermatozoa from
the immature and less motile fraction.
The fourth part of the study was aimed at investigating the effects of the cytokines,
tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), on human sperm
motility, viability, acrosome reaction and NO production. The study shows that TNF-α
and IL-6 significantly reduced motility parameters and acrosome reaction in a dose4
and time-dependent manner. These cytokines were also shown to significantly
increase NO production from human spermatozoa. The decreased motility
parameters induced by these cytokines could be attributed to their ability to induce
excessive NO production. It is not yet clear how they inhibit spermatozoa to undergo
the acrosome reaction.
The fifth part of the study was to investigate the expression and localization of
glucose transporter 8 (GLUT8) in human spermatozoa. This study shows that GLUT8
is constitutively expressed and located in the midpiece region of the human
spermatozoa. The study also showed that stimulating spermatozoa with insulin led to
an increase in GLUT8 expression as well as translocation to the acrosomal region.
In the last part of the study we wanted to investigate why the increase in NO
generation by spermatozoa due to insulin and leptin stimulation is accompanied with
increased sperm function whereas NO increased due to TNF-α and IL-6 stimulation
is accompanied with decreased sperm function. We observed that TNF-α and IL-6
not only increased NO production but also ROS production. This study speculates
that the decrease in sperm motility and acrosome reaction when TNF-α and IL-6
were administered was due to the concomitant high increase in NO and ROS they
induced.
In conclusion, this study has established in vitro beneficial effects of insulin and leptin
in normozoospermic and asthernozoospermic human sperm function. These
hormones influence sperm function via the PI3K signalling pathway in two ways.
Firstly, by increasing GLUT8 expression and translocation thereby possibly
increasing glucose uptake and metabolism and secondly, by increasing NO
production. The study has also established that TNF-α and IL-6 have detrimental
effects on human spermatozoa in a dose and time dependent manner. These effects
are mediated via their ability to stimulate both NO and ROS production in human
spermatozoa. This study reports that GLUT8 is expressed in the midpiece region of
human spermatozoa and that insulin stimulation upgrades its expression and leads to
its translocation to the acrosomal region. / AFRIKAANSE OPSOMMING: Oor die afgelope jare was daar `n toename in obesiteit en diabetes mellitus (DM).
Hierdie toestande word reeds vir ’n geruime tyd geassosieer met onvrugbaarheid.
Obesiteit word gekenmerk deur verhoogde sirkulerende vlakke van leptiene en
sitokiene sowel as insulien weerstandigheid. Tipe I DM word geassosieer met lae of
geen insulien terwyl Tipe II DM gekenmerk word deur insulien weerstandigheid. Soos
wat die voorkoms van obesiteit en DM toeneem, is dit waarskynlik dat die insidensie
van onvrugbaarheid wat met hierdie patologiese toestande geassosieer word,
gevolglik ook sal toeneem. Die effek van insulien en leptien op die manlike
voortplantingsfunksie is alreeds aangetoon op endokriene en spermatogenese vlak,
maar hul effekte op sellulêre vlak van menslike geëjakuleerde spermatosoë is nog
onduidelik.
Die studie vertoon data oor die rol van insulien en leptien op die menslike
geëjakuleerde spermatosoë en hul interaksie met sitokiene en stikstofoksied (NO). In
die eerste gedeelte van die studie, het ons ’n toepaslike konsentrasie van insulien en
leptien bepaal wat aan menslike spermatosoë in vitro toegedien kan word. Glukose
konsentrasies van 5,6 mM is bepaal as die gepaste konsentrasie om aan menslike
spermatosoë toe te dien, omdat dit beter resultate tot gevolg het; verder is dit
vergelykbaar met fisiologiese glukose vlakke in die bloed van `n vastende persoon.
Insulien en leptien konsentrasies is op 10 μIU en 10 nm onderskeidelik vasgestel,
aangesien dit tot beter resultate gelei het, en omdat dit vergelykbaar was met
insulien en leptien vlakke wat reeds voorheen in menslike geëjakuleerde
spermatosoë gemeet is. Dit was gevolg deur `n ondersoek na die insulien
seintransduksie pad en sy voordelige effekte op menslike spermatosoë funksie.
Endogene insulien afskeiding deur menslike geëjakuleerde spermatosoë was deur
nifedipien geïnhibeer en sy reseptor tirosien fosforilasie effekte was deur erbstatin
geïnhibeer terwyl fosfatidielinositol 3-kinase (PI3K) fosforilasie deur wortmannin
geïnhibeer is. Eksogene insulien toediening het menslike sperm-motiliteit parameters
betekenisvol laat toeneem asook die vermoë van sperme om die akrosoomreaksie te
ondergaan. Die inhibisie van endogene insulien afskeiding deur spermatosoë sowel
as die inhibisie van die insulien reseptor substraat (IRS) tirosien fosforilasie het die
motiliteit parameters en die akrosoomreaksievermoë van spermatosoë verlaag.
Die studie het ook die effekte van insulien en leptien op menslike sperm-motiliteit,
-lewensvatbaarheid, -akrosoomreaksie en -NO produksie nagevors. Beide insulien
en leptien het sperm-motiliteit parameters, -akrosoomreaksie en -NO produksie
betekenisvol verhoog. NO produksie is deur insulien en leptien via PI3K
seintransduksie geïnduseer, soos bewys deur die verlaging waargeneem in NO
vlakke toe PI3K aktiwiteit deur wortmannin geïnhibeer was. Om vas te stel of insulien
en leptien die motiliteit parameters van asthenozoospermiese en teratozoospermiese
spermatosoë kon verbeter, het ons spermatosoë in twee fraksies met ’n dubbel
digtheid gradiënt geskei. Die gradiënt sisteem was daartoe instaat om die
spermatosoë in ’n onvolwasse, (morfologies abnormaal en minder motiel - soortgelyk
aan dié van asthenozoospermiese en teratozoospermiese pasiënte), sowel as ’n
volwasse meer motiele fraksie te skei. Insulien en leptien het die motiliteit parameters
van spermatosoë van die onvolwasse en minder motiele fraksie verhoog.
Die vierde gedeelte van die studie was daarop gemik om die effekte van die sitokiene
tumor nekrose faktor alfa (TNF-α) en interleukin-6 (IL-6) op menslike sperm-motiliteit,
-lewensvatbaarheid, -akrosoomreaksie en -NO produksie, te ondersoek. Die studie
het getoon dat TNF-α en IL-6 motiliteit parameters en akrosoomreaksie in ’n tyd- en
dosis-afhanklike wyse betekenisvol verlaag het. Hierdie sitokiene was ook in staat
om NO produksie in menslike spermatosoë te verhoog. Die verlaging in motiliteit
parameters wat deur hierdie sitokiene geïnduseer is, kan toegeskryf word aan hul
vermoë om die produksie van oormatige hoeveelhede NO te stimuleer. Dit is nog nie
duidelik hoe hulle die akrosoomreaksie in spermatosoë kan inhibeer nie.
Die vyfde gedeelte van die studie het dit ten doel gehad om die uitdrukking en
lokalisering van die glukose transporter 8 (GLUT8) in menslike spermatosoë te
ondersoek. Hierdie studie kon aantoon dat GLUT8 konstitutief uitgedruk is en in die
middelstuk van die menslike spermatosoë voorkom. Die studie bewys ook dat
stimulering van die spermatosoë met insulien tot `n toename in GLUT8 uitdrukking
sowel as translokasie na die akrosomale area, lei.
In die finale gedeelte van die studie wou ons ondersoek waarom die toename in NO
produksie in spermatosoë (as gevolg van insulien en leptien stimulasie) deur `n
toename in spermfunksie gekenmerk word, terwyl die toename in NO produksie (as
gevolg van TNF-α en IL-6 stimulasie) deur ’n afname in spermfunksie gekenmerk
word. Ons het waargeneem dat TNF-α en IL-6 nie alleen NO produksie nie, maar ook
reaktiewe suurstof spesies (ROS) produksie verhoog het. Ons vermoed dat die
afname in sperm motiliteit en akrosoomreaksie met TNF-α en IL-6 toediening, die
gevolg van die gelyktydige verhoging in NO en ROS was.
In gevolgtrekking kan ons sê dat hierdie studie die voordelige in vitro effekte van
insulien en leptien op asthenozoospermiese en teratozoospermiese menslike
spermfunksie aangetoon het. Hierdie hormone beïnvloed spermfunksie via die PI3K
seintransduksie pad op twee maniere. Eerstens, deur `n toename in GLUT8
uitdrukking en translokasie, met die gevolg dat glukose opname en metabolisme
moontlik verhoog is, en tweedens, deur die toename in NO produksie. Die studie het
ook vasgestel dat TNF-α en IL-6 nadelige effekte op menslike spermatosoë in `n
dosis- en tyd-afhanklike wyse het. Hierdie effekte vind plaas a.g.v. hul vermoë om
beide NO en ROS produksie in menslike spermatosoë te induseer. Die studie toon
aan dat GLUT8 uitdrukking in die middelstuk van die menslike spermatosoon
voorkom en dat insulien stimulasie GLUT8 uitdrukking opreguleer en tot translokasie
na die akrosomale area lei.
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The impact of developmental stress on the functioning and vulnerability of CNS neuronsPienaar, Ilse-Sanet 12 1900 (has links)
Thesis (PhD (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / The overall objective of this thesis is to provide additional data to assist clinicians and
experimental neurologists alike in the quest for better understanding, more accurately
diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD).
The general theme of the thesis is the interaction between certain environmental stimuli,
including the exposure to adverse events during early central nervous system (CNS)
development and the manifestation of elements of neurodegeneration, whether by means of
neurochemical changes or expressed as a dysfunctional voluntary motor system.
The first chapter provides a general introduction to the research theme of the thesis. This
includes, in particular, a discussion on current understanding concerning the etiology and
clinical profile of PD, the relative contribution made by genetic factors compared to
environmental ones, and current treatment strategies for treating the disease. Mention is also
made of the failure of these therapeutic applications for reversing or protecting against the
disease, due to the side-effects associated with them. The material covered in chapter 1
provides the basis for the more complete discussion concerning these various aspects,
contained in the chapters to follow.
The overall aim was also to characterise the effects of commonly used toxin-induced animal
models of PD, and the extent of vulnerability that the CNS displays towards them. The
destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points
along the nigrostriatal tract is used extensively to model PD pathology in rats and is an
established animal model of the disease. However, mature or even aged animals are mainly
used in these studies, while the effects that the toxin might have on the developing CNS remain
unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the
young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a
unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial
forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a
direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum.
Although both lesion types were used, the MFB model is considered a more accurate portrayal
of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of
the disease. The different lesions’ effects on motor function were determined by observing
animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of
a cylindrical enclosure. Following the final behavioral assessment, the concentration of
dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using
4
HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The
HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the
lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment,
there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic
terminals that may be sufficient for maintaining relatively high extracellular and synaptic
concentrations of DA. However, since substantial changes in motor-function were observed, it is
suggested that the capacity of the remaining dopaminergic neurons to respond to increased
functional demands may be limited. In addition, the behavioral results indicate that the distinct
indices relating to different functional deficits depend on the lesioning of anatomically distinct
structures along the nigrostrial tract.
It has long been known that far fewer women are diagnosed with PD than men are. This
seeming protection offered to females against degenerative disease of the CNS may relate to
estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly
defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United
States alone, the aim of chapter 2 was to examine the evidence for a possible relationship
between PD and the female reproductive hormone estrogen. A review of the current literature
available on the topic was performed by consulting Medline, and by performing a search of the
case-reports contained within the World Health Organization’s (WHO) International Drug
Monitoring database, for possible PD-related symptoms that may arise from estrogen
replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen
protects women from obtaining the disease, or at least some features of it. Intensive research
efforts are called for, with sufficient power to establish the relationship between ERT and the
onset and development of parkinsonism. Chapter 3 reports on the results obtained from an
experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher
dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14
days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6-
OHDA the female rats were less impaired than males in making adjustment steps in response
to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine
hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender
differences in cell survival factors and/or other promoters of neuroplasticity may have
contributed to the beneficial outcome seen in the females. For example, nerve growth factor
(NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It
is unclear whether gonadal steroids are involved, and, if so, whether female hormones are
protective or whether male hormones are prodegenerative. Determining the mechanisms for the
improved outcome seen in the young female rats may lead to potential treatment strategies
against PD.
5
Many studies have shown that early life stress may lead to impaired brain development, and
may be a risk factor for developing psychiatric diseases, including clinical depression. However,
few studies have investigated the impact that early stress may have on the onset and
development of neurodegenerative disorders such as PD. The study reported on in chapter 5
conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well
characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6-
OHDA. The combined effects of these models on motor deficits and brain protein levels were
investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion
with a battery of tests that are sensitive to the degree of DA loss sustained. The results
show that animals that had been subjected to MS display poorer performance in the vibrissae
and single-limb akinesia test compared to non-MS control animals (that had also been
subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH
staining in MS rats compared to non-MS ones. The results from this study therefore suggest
that exposure to adverse experiences during the early stages of life may contribute towards
making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring
during mature stages of life. Therefore, taken together, early exposure to stress may predispose
an individual towards the onset and development of neurodegenerative disease, which
especially becomes a threat during the later stages of adult life.
Moreover, within the framework of these characteristics, the capacity of a widely-used
pharmacological agent (statins) was tested for possible future therapeutic application in PD
(chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests
that it may associate with defective activity of complex I of the mitochondrial electron transport
chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased
oxygen free radical production, depressed antioxidant enzyme activities, and greater
susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for
lowering cholesterol levels in patients who display elevated concentrations of low-density
lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on
motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I
inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with
simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor
function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb
placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally
lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the
vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified
concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2-
6
Dimensional electrophoresis (2-DE) with subsequent identification of the spots using
electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was
performed and the results BLAST-searched using bio-informatics tools for naming the identified
peptides. The motor test results indicate that while unilateral rotenone causes behavioral
asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced
ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in
protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly
classified according to their cellular function into 6 categories, with the majority involved in
energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated
techniques and non-biased ability to quantify proteins, provides a methodology with which to
study the changes in neurons associated with neurodegeneration. As an emerging tool for
establishing disease-associated protein profiles, it also generates a greater understanding as to
how these proteins interact and undergo post-translational modifications. Furthermore, due to
the advances made in bioInformatics, insight is created concerning their functional
characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss
major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped
that the application of this technology will lead towards a presymptomatic diagnosis of PD, and
the identification of risk factors and new therapeutic targets at which pharmacological
intervention can be aimed.
The final chapter (chapter 8) provides a retrospective look at the academic work that had
been performed for the purpose of this thesis, recaps on the main findings, and also highlights
certain aspects of the project and provides relevant suggestions for future research. Lastly, the
appendix provides a detailed overview of the methods followed for the experiments described in
this thesis. It provides not only a comprehensive description of the techniques that had been
followed, but provides information concerning the care taken with the animals (i.e. post-surgery)
in order to control for the potential influence of experimental variables on the results.
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The potential of exercise to reverse stress induced abnormalities in the rat brainMarais, Lelanie 03 1900 (has links)
Thesis (PhD (Biomedical Sciences. Medical Physiology.))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Adverse experiences during early life causes alterations in the development of the
central nervous system structures that may result in abnormal functioning of the
brain. It is well known that, in humans, adverse early-life experiences such as social
separation, deprivation, maternal neglect and abuse increase the risk of developing
psychiatric disorders, such as depression, later in life. We used maternal separation
in the rat as a model for early life stress to firstly determine how different brain
systems are dysregulated by this stressful experience and additional chronic or acute
stress during adulthood. Rat pups were separated from their mothers on postnatal
day 2-14 for 3 hours per day while control rats were normally reared. The behavior,
stress response, neurotrophin, apoptotic marker and serotonin levels in the ventral hippocampus, striatum and frontal cortex were measured during adulthood. A
different group of maternally separated rats were allowed chronic voluntary exercise
and similar measurements were done to determine whether exercise was able to
normalize the deficits caused by early life stress. Differentially expressed cytosolic
proteins of the ventral hippocampus of maternally separated rats versus normally
reared rats were also identified. Protein expression levels of maternally separated
rats that received chronic voluntary exercise or escitalopram treatment were
subsequently determined to unravel the mechanism of therapeutic action for these
two interventions. We found that maternal separation increased the baseline
corticosterone response of rats and induced a blunted adrenocorticotropin hormone
after acute restraint stress. Baseline neurotrophin levels were significantly
decreased in the ventral hippocampus. Maternal separation followed by chronic
restraint stress during adulthood resulted in increased depressive-like behavior
compared to control rats. Maternal separation alone or followed by acute restraint stress during adulthood induced changes in apoptotic marker expression in the
striatum and frontal cortex. In rats subjected to maternal separation and chronic
restraint stress during adulthood, we found that chronic voluntary exercise decreased
their depressive-like behavior and increased brain derived neurotrophin levels in the
striatum. Serotonin levels were not affected by maternal separation, but chronic
voluntary exercise increased serotonin in the ventral hippocampus of normally reared
rats. Maternal separation induced a number of changes in the expression of
cytosolic proteins and these stress-induced changes were identified in proteins
relating to cytoskeletal structure, neuroplasticity, oxidative stress, energy metabolism,
protein metabolism, and cell signaling. Chronic voluntary exercise was able to
restore the expression levels of a number of proteins affected by maternal separation
that increased the risk for neuronal death. When comparing the efficacy of exercise
to that of escitalopram treatment it was evident that, in contrast to exercise,
escitalopram targets a different subset of proteins affected by maternal separation,
except for a few involved in energy metabolism pathways and neuroprotection. In
this study we have shown that chronic voluntary exercise has therapeutic effects in
maternally separated rats, decreasing depressive-like behavior, increasing
neurotrophin expression and restoring cytosolic protein expression that were dysregulated by early life stress. / AFRIKAANSE OPSOMMING: Negatiewe stresvolle ervarings gedurende die vroeë stadium van ‘n mens se lewe
veroorsaak veranderinge in die ontwikkeling van breinstrukture en het ‘n nadelige
uitwerking op die funksionering van die brein. Dit is bekend dat stresvolle ervarings
in kinders, byvoorbeeld sosiale afsondering, verwaarlosing en mishandeling, die
risiko vir die ontwikkeling van psigiatriese steurings soos depressie gedurende
volwassenheid kan verhoog. In hierdie studie gebruik ons moederlike skeiding van
neonatale rotte as ‘n model vir vroeë lewensstres om te bepaal hoe dit verskillende
sisteme in die brein negatief beinvloed, en dan ook die effek van addisionele
kroniese of akute stres gedurende volwassenheid. Die neonatale rotte is
weggeneem van hulle moeders af vanaf dag 2 tot 14 vir 3 ure elke dag terwyl
kontrole rotte by hulle moeders gebly het. Die gedrag, stres respons, neurotrofiene,
apoptotiese merkers en serotonien vlakke is gemeet in die ventrale hippokampus,
frontale korteks en striatum gedurende volwassenheid. Rotte wat van hulle moeders
geskei is, is dan toegelaat om vir ses weke in wiele te hardloop om te bepaal of
kroniese vrywillige oefening die negatiewe effekte wat veroorsaak is deur stres kan
ophef. ‘n Bepaling van sitosoliese proteien uitdrukking in die ventrale hippokampus
is ook gedoen om die uitwerking van moederlike skeiding op proteienvlak vas te stel.
Hierdie protein data is dan vergelyk met die van gestresde rotte wat kroniese
oefening of escitalopram behandeling ontvang het om die meganisme van werking van beide behandelings te bepaal. Ons het gevind dat moederlike skeiding die
rustende kortikosteroon vlakke van rotte verhoog terwyl dit adrenokortikotropien
vlakke na akute stres inhibeer. Moederlike skeiding het ook die neurotrofien vlakke
in die ventrale hippokampus verlaag en addisionele kroniese stres gedurende
volwassenheid het ‘n verhoging in depressie-agtige gedrag veroorsaak. Moederlike skeiding alleen, sowel as gevolg deur akute stress gedurende volwassenheid het ook
veranderinge in die uitdrukking van apoptotiese merkers in die striatum en frontale
korteks veroorsaak. Kroniese vrywillige oefening na moederlike skeiding en
addisionele stres gedurende volwassenheid kon depressie-agtige gedrag verlaag en
neurotrofienvlakke in die striatum verhoog. Serotonien vlakke was nie beinvloed
deur moederlike skeiding nie, maar oefening in kontrole rotte het serotonien verhoog
in die ventrale hippokampus. Moederlike skeiding het heelwat veranderinge in die
uitdrukking van sitosoliese proteiene van die ventrale hippokampus veroorsaak wat
ingedeel kan word in die volgende funksionele kategorieë: sitoskelet,
neuroplastisiteit, oksidatiewe stres, energiemetabolisme, proteinmetabolisme en
seintransduksie. Oefening kon die uitdrukking van verskeie stres-geïnduseerde
veranderinge in proteiene weer herstel terwyl dit wou bleik asof escitalopram se
meganisme van werking op ‘n ander vlak geskied. Ons bevindinge bewys dat
kroniese vrywillige oefening ‘n goeie behandeling is na vroeë lewenstres en dat dit
depressiewe gedrag verminder, neurotrofien vlakke verhoog en sitosoliese proteien skeiding alleen, sowel as gevolg deur akute stress gedurende volwassenheid het ook
veranderinge in die uitdrukking van apoptotiese merkers in die striatum en frontale
korteks veroorsaak. Kroniese vrywillige oefening na moederlike skeiding en
addisionele stres gedurende volwassenheid kon depressie-agtige gedrag verlaag en
neurotrofienvlakke in die striatum verhoog. Serotonien vlakke was nie beinvloed
deur moederlike skeiding nie, maar oefening in kontrole rotte het serotonien verhoog
in die ventrale hippokampus. Moederlike skeiding het heelwat veranderinge in die
uitdrukking van sitosoliese proteiene van die ventrale hippokampus veroorsaak wat
ingedeel kan word in die volgende funksionele kategorieë: sitoskelet,
neuroplastisiteit, oksidatiewe stres, energiemetabolisme, proteinmetabolisme en
seintransduksie. Oefening kon die uitdrukking van verskeie stres-geïnduseerde
veranderinge in proteiene weer herstel terwyl dit wou bleik asof escitalopram se
meganisme van werking op ‘n ander vlak geskied. Ons bevindinge bewys dat
kroniese vrywillige oefening ‘n goeie behandeling is na vroeë lewenstres en dat dit
depressiewe gedrag verminder, neurotrofien vlakke verhoog en sitosoliese proteien vlakke kan herstel.
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The mechanism of pharmacological preconditioning of rat myocardium with beta-adrenergic agonistsSalie, Ruduwaan 03 1900 (has links)
Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The Mechanism of -adrenergic preconditioning ( -PC)
Ischaemic preconditioning (IPC), a potent endogenous protective intervention against myocardial
ischaemia, is induced by exposure of the heart to repetitive short episodes of ischaemia and
reperfusion. The protective effects of this phenomenon have been demonstrated to be mediated by
release of autocoids such as adenosine, opioids and bradykinin. Release of endogenous
catecholamines and activation of the beta-adrenergic receptors (b-AR) have also been shown to be
involved in ischaemic preconditioning. However, the exact mechanism whereby activation of the -
adrenergic signal transduction pathway leads to cardioprotection, is still unknown.
In view of the above, the aims of the present study were to evaluate:
(i) the respective roles of the 1-, 2- and 3-AR receptors as well as the contribution of Gi
protein and PKA to -adrenergic preconditioning,
(ii) the role of the prosurvival kinases, PKB/Akt and ERK 44/p42 MAPKinase in -drenergic
preconditioning,
(iii) whether b-AR stimulation protect via ischaemia and the formation of adenosine; the
respective roles of the A1-, A2-, A3-adenosine receptors as well as the involvement of the
PI3-K/PKB/Akt and ERKp44/p42 signal transduction pathways, in the cardioprotective
phenomemon of -adrenergic preconditioning and
(iv) the contribution of the mitochondrial KATP channels (mKATP), reactive oxygen species and NO
to the mechanism of -AR-induced cardioprotection.
Methods: Isolated perfused rat hearts were subjected to 35 min regional ischaemia (RI) and
reperfusion. Infarct size (IS) was determined using tetrazolium staining (TTC) and data were
analyzed with ANOVA. Hearts were preconditioned with 5 min isoproterenol 0.1 μM ( 1/ 2-AR
agonist), or formoterol 1 nM ( 2-AR agonist) or BRL 37344 1 μM ( 3-AR agonist) followed by 5
min reperfusion. The roles of the 1-, 2- and 3-ARs as well as NO were explored by using the
selective antagonists CGP-20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) and NOS
inhibitors L-NAME (50 μM) or LNNA (50 μM) respectively. Involvement of ROS and the mK+
ATP
channels was studied by administration of N-acetyl cysteine (NAC, 300 μM) and the mitK+
ATP
iv
channel blocker 5-HD (100 μM) during the triggering phase. The role of PKA and PI3-K/Akt was
investigated by the administration of the blockers Rp-8-CPT-cAMPs (16 μM) and wortmannin (100
nM) respectively, prior to RI or at the onset of reperfusion. Pertussis toxin (PTX), 30 μg kg-1 was
administered i.p., 48 h prior to experimentation.
The role of adenosine and the adenosine A1, A3, A2A and A2B receptors was studied by using
adenosine deaminase and the selective antagonists DPCPX (1 μM), MRS 1191(1 μM), ZM241385
(1 μM) and MRS1754 (1 μM). Activation of PKB/Akt and ERKp44/p42 was determined by
Western blot.
Results: Infarct sizes of hearts preconditioned with isoproterenol of formoterol were significantly
smaller compared to those of non-preconditioned hearts. This was associated with an improvement
in postischaemic mechanical performance. However the 3-AR agonist BRL37344 could not reduce
infarct size. The 1- and 2-AR blockers CGP-20712A and ICI-118551 completely abolished the
isoproterenol-induced reduction in infarct size and improvement in mechanical recovery, while the
3-AR blocker was without effect.
Both Rp-8-CPT-cAMPs and wortmannin significantly increased infarct size when administered
before 1/ 2-AR preconditioning or at the onset of reperfusion while it reduced mechanical
recovery during reperfusion. PTX pretreatment had no significant effect on the reduction in infarct
size induced by 1/ 2-AR or 2-AR preconditioning, however it reduced mechanical recovery in the
latter. The NOS inhibitors had no effect on the reduction in infarct size induced by 1/ 2-AR
preconditioning, but depressed mechanical function during reperfusion.
The significant reduction in infarct size by 1/ 2-PC, was associated with activation of ERKp44/p42
and PKB/Akt during the triggering phase, as well as during reperfusion. DPCPX (A1-AdoR
antagonist) had no effect on the 1/ 2-PC-induced reduced infarct size or ERK p44/p42 and PKB
activation.
A2A-AdoR, but not A2b-AdoR, blockade during the trigger phase abolished the reduction in infarct
size of 1/ 2-PC. Both antagonists significantly reduced ERK and PKB activation in the trigger
phase. In addition, when applied at the onset of reperfusion they significantly reduced ERK p44 /
v
p42 MAPK and PKB/Akt activation to an even greater extent. MRS-1191 (A3-AdoR antagonist)
blocked 1/ 2-PC when applied prior to index ischaemia or when added during early reperfusion,
significantly inhibiting both ERK p44 and PKB activation.
Cardioprotection of 1/ 2-PC was abolished by inhibition of ROS generation with NAC in the
triggering phase as well as at the start of reperfusion. However, the mitoK+
ATP channel blocker 5-
HD was without effect.
Conclusions: Protection afforded by an acute transient stimulation of the -ARs, depends on the
activation of both 1-AR and 2-ARs but not the 3-AR. PKA as well as PI3-K activation prior to
sustained ischemia and at the onset of reperfusion were essential for cardioprotection. With
functional recovery as endpoint, it appears that NO is involved in 1/ 2-AR preconditioning, while
the Gi protein may play a role in 2-AR preconditioning.
The production of endogenous adenosine induced by transient b1/b2 stimulation of the isolated rat
heart is involved in b−AR preconditioning. Cardioprotection was shown not to be dependent on the
A1AdoR while activation of the A3-AdoR occurs during both the triggering and mediation phases.
Both the adenosine A2A and, to a lesser extent, the adenosine A2B receptors participate in the
triggering phase of b1/b2-PC. Generation of ROS during the triggering and reperfusion phases is
involved in eliciting protection, but no role for the mKATP channels could be demonstrated. Finally,
activation of the RISK pathway (PKB/Akt and ERKp44/p42) during the triggering phase is a
prerequisite for protection. In addition, cardioprotection by b-AR is characterized by activation of
the RISK pathway during reperfusion. / AFRIKAANSE OPSOMMING: Iskemiese prekondisionering (IPC) is ‘n kragtige endogene beskerming teen miokardiale iskemie,
wat deur blootstelling van die hart aan kort opeenvolgende episodes van iskemie en herperfusie,
ontlok word. Hierdie beskerming word medieer deur vrystelling van outakoïede soos adenosine,
opioïede en bradikinien. Vrystelling van endogene katekolamiene en aktivering van die betaadrenerge
reseptore (b-AR) is bewys om ook by hierdie proses betrokke te wees. Die presiese
meganismes waardeur aktivering van die -adrenerge seintransduksiepad tot miokardiale
beskerming lei, is nog onbekend.
In die lig van bogenoemde, was die doel van die huidige studie om die volgende te evalueer: (i) die
onderskeie rolle van die b1-, b2- en b3-AR sowel as die bydrae van die Gi proteïen en PKA in b-
adrenerge prekondisionering, (ii) of b-AR stimulasie beskerming ontlok via iskemie en vorming van
adenosien, die onderskeie rolle van die A1-, A2-, A3-adenosien reseptore (AdoRs) sowel as die PI3-
K/PKB/Akt en ERKp44/p42 seintransduksie paaie, (iv) die mitochondriale KATP (mKATP) kanale,
vry suurstof radikale en NO in b−AR prekondisionering.
Metodes: Geïsoleerde, geperfuseerde rotharte is aan 35 minute streeksiskemie en herperfusie
onderwerp. Infarktgrootte (IS) is deur die tetrazolium (TTC)-kleuringsmetode bepaal. Data is met
behulp van ANOVA analiseer. Harte is geprekondisioneer vir 5 min met isoproterenol 0.1 μM
( 1/ 2-AR agonist), of formoterol 1 nM ( 2-AR agonist) of BRL 37344 1 μM ( 3-AR agonist),
gevolg deur 5 min herperfusie, voor streeksiskemie. Die belang van die 1-, 2- en 3-ARs sowel
as NO is ondersoek, deur onderskeidelik gebruik te maak van selektiewe antagoniste nl CGP-
20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) en NOS inhibitore L-NAME (50μM)
of LNNA (50μM). Die rol van die mK+
ATP kanale en ROS is bepaal deur die toediening van die
mK+
ATP kanaal blokker 5-HD (100 μM) en die vrye-radikaal opruimer, N-asetiel cysteine (NAC,
300 μM). Die belang van PKA en PI3-K/Akt is bepaal deur toediening van die PKA blokker Rp-8-
CPT-cAMPs (16μM) en wortmannin (100nM) respektiewelik. Pertussis toxin (PTX), 30 μg kg-1 is
i.p toegedien, 48 uur voor eksperimentasie.
vii
Die rol van adenosien en die adenosien A1, A2A, A2B en A3 reseptore is bestudeer, deur gebruik te
maak van adenosien deaminase en die selektiewe antagoniste DPCPX (1 μM), MRS 1191(1 μM),
ZM241385 (1 μM) and MRS1754 (1 μM),repektiewelik. Die middels is deurgaans toegedien tydens
die prekondisioneringsprotokol (“snellerfase”) of tydens vroeë herperfusie. Aktivering van PKB/Akt
en ERK p44/p42 is deur Western blot analise bepaal.
Resultate: Infarktgrootte van harte wat geprekondisioneer is met of isoproterenol ( 1/ 2-PC) of
formoterol ( 2-PC), was beduidend kleiner as díe van ongeprekondisioneerde harte. Dit is
geassosieer met ‘n toename in postiskemiese meganiese herstel. Die 3-AR agonis BRL37344 ( 3-
PC) het egter geen effek op infarktgrootte gehad nie. Die selektiewe 1- en 2-AR blokkers CGP-
20712A en ICI-118551 het die afname in infarktgrootte heeltemal opgehef, asook die verbetering in
meganiese herstel tydens herperfusie terwyl die 3-AR blokker geen effek getoon het nie. Beide Rp-
8-CPT-cAMPs en wortmannin het infarktgrootte beduidend vergroot en meganiese herstel
beduidend verlaag, wanneer dit net voor 1/ 2-prekondisionering of tydens die begin van
herperfusie toegedien is. PTX voorafbehandeling het geen beduidende effek op die vermindering
van infarktgrootte (geïnduseer deur 1/ 2-PC of 2-PC) gehad nie. Meganiese herstel is egter
verminder in die geval van 2-PC. Die NOS inhibitore het geen effek op die vermindering in
infarktgrootte geïnduseer deur b1/b2 gehad nie, maar het ook meganiese herstel onderdruk.
Die beduidende afname in infarktgrootte deur b1/b2 prekondisionering is gekenmerk deur
aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase. Soortgelyke aktivering van hierdie
kinases is ook tydens herperfusie van b-AR geprekondisioneerde harte waargeneem.
DPCPX (A1-AdoR antagonis) het geen effek op die infarkt-verminderde effek van 1/ 2-
prekondisionering of op ERK p44/p42 en PKB aktivering gehad nie. A2A-AdoR, maar nie A2b –
AdoR, blokkade tydens die snellerfase, het die effek van b-AR prekondisionering op infarktgroottee
opgehef. Beide antagoniste het die aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase
onderdruk. Wanneer toegedien tydens herperfusie, het dit die aktivering van hierdie kinases tot ‘n
groter mate onderdruk. MRS-1191 (A3-AdoR antagonis) het infarktgrootte beduidend verhoog en
1/ 2-prekondisionering geblokkeer, beide wanneer dit voor indeks-iskemie toegedien is of tydens
vroeë herperfusie, tesame met inhibisie van PKB en ERK p44/p44 aktivering.
viii
Die kardiobeskerming van 1/ 2-prekondisionering is opgehef deur middel van opruiming van vry
suurstof radikale deur NAC in die snellerfase sowel as aan die begin van herperfusie. Die mK+
ATP
kanaal blokker 5-HD het geen effek op b-AR prekondisionering gehad nie.
Gevolgtrekking: Kardiobeskerming teweeggebring deur ‘n kort periode van stimulasie van die -
ARs, is afhanklik van die aktivering van beide 1-AR en 2-ARs, maar nie 3-AR nie. PKA sowel
as PI3-K aktivering, net voor volgehoue iskemie en tydens vroeë herperfusie, is aangedui om
noodsaaklik vir 1/ 2-AR prekondisionering te wees. Waar funksionele herstel as eindpunt gebruik
is, blyk dit dat NO wel betrokke is by 1/ 2-AR prekondisionering, terwyl die Gi protein ‘n rol mag
speel in 2-AR prekondisionering.
Vorming van endogene adenosien tydens b-adrenerge stimulasie is betrokke by b-AR
prekondisionering. Hierdie beskerming is nie van die A1-AdoR afhanklik nie, maar aktivering van
die A3-AdoR is nodig tydens beide die sneller en herperfusie fases. Beide die A2A-AdoR, en tot ‘n
mindere mate die A2B–AdoR, is ook betrokke by die snellerfase. Vorming van vry suurstof radikale
is nodig vir b-AR prekondisionering, nterwyl die mKATP kanale nie betrokke is nie. Ten slotte,
aktivering van die RISK seintransduksiepad (ERKp42/p44 en PKB/Akt) tydens die snellerfase is ‘n
voorvereiste vir die ontlokking van beskerming. Daarbenewens word b-AR prekondisionering
gekarakteriseer deur aktivering van hierdie pad tydens herperfusie. / South African Medical Research Council / University of Stellenbosch
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Ischaemic preconditioning : an investigation of the patterns of kinase activation and protein expression profiles during reperfusion in the rat heartHattingh, Susanna Maria (Suzel) 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of death
worldwide with 3.8 million men and 3.4 million women dying globally each year.
Although existing myocardial reperfusion strategies such as thrombolysis and
percutaneous coronary intervention (PCI), if applied in a timely manner, limit
myocardial infarct size, the mortality and morbidity remains significantly high.
Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardial
ischaemia/reperfusion injury through cardioprotective signaling pathways which is
recruited at the time of myocardial reperfusion, thereby improving clinical
outcomes in patients with coronary artery disease.
Ischaemic preconditioning is a phenomenon whereby short intermittent episodes
of coronary occlusion followed by reperfusion protect the myocardium against a
subsequent period of sustained ischaemia. This protection is reflected in the
limitation of infarct size and improved functional recovery of the ischaemic heart
during reperfusion. Despite intensive research efforts, the promise of an effective
cardioprotective strategy using the endogenous protective mechanisms of the
heart which underlies IPC, has not yet been materialized. Although progress has
been made in terms of signaling mechanisms in the preconditioned heart, the
identification of the myocardial reperfusion phase as the critical “window” for
cardioprotection, requires the elucidation of the signal transduction pathways
during the reperfusion phase after IPC.
In view of the above, the aims of the present study were to investigate:
i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPC
during early and late reperfusion
ii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70
(HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB in
the context of IPC during early reperfusion
iii. the involvement of autophagy and apoptosis during early and late
reperfusion after IPC iv. the correlation of the protein kinases with the hemodynamic parameters of
the heart
v. the mechanism of IPC by means of two-dimensional (2D) proteomics
Methods: The isolated perfused working rat heart model was used with
functional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 min
global ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25
min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion when
hearts were snap-frozen for western blotting analysis. Alternatively, hearts were
reperfused for 30 min to record hemodynamic parameters and measure
functional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30
min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or
30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC hearts
were reperfused for 30 min to serve as control for the 30 min reperfused IPC
group. Activation of the protein kinases was determined by western blotting
analysis.
For the proteomic study mitochondrial and cytosolic proteins were isolated from
heart tissue and separated in the first dimension by isoelectric focusing, followed
by separation in the second dimension by two dimensional gel electrophoresis.
The PD Quest software programme was used to identify significantly expressed
protein spots. Protein spots of interest were excised and subjected to in-gel
digestion and the resulting peptides were analysed by mass spectrometry.
Proteins were identified by Mascot and the Swiss Prot database.
Results: Western blotting analysis demonstrated that the RISK pathway and p38
MAPK are activated very early in reperfusion, but the activation is not sustained
during the reperfusion period. Autophagy is also upregulated during this early
reperfusion phase; it is attenuated in the middle reperfusion phase and increase
for a second peak of upregulation in the late reperfusion phase. In addition, we
identified CAMKII as a novel marker of functional recovery in IPC after
reperfusion. The proteomic analysis identified twenty differentially expressed mitochondrial
and thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins were
directly related to cardiac metabolism.
Conclusion: Activation of the majority of the protein kinases investigated in the
present study is associated with the hemodynamic parameters of the heart
instead of functional recovery. Results indicated that the variable signaling
patterns could be attributed to differences in heart rate and the effect thereof
(ejection fraction, minimum and maximum rate of contraction), as a result of
sympathetic stimulation due to psychological stress in the animals before
slaughtering. Proteomics results demonstrated that IPC hearts which failed after
ischaemia /reperfusion are metabolically compromised and “worse off” compared
to non-IPC hearts. / AFRIKAANSE OPSOMMING: Inleiding: Koronêre hartsiekte is die vernaamste oorsaak van sterftes wêreldwyd
met 3.8 miljoen mans en 3.4 miljoen vrouens wat jaarliks sterf. Alhoewel
bestaande miokardiale herperfusie strategieë soos trombolise en perkutane
koronêre intervensie (PKI), wanneer betyds toegepas, miokardiale infarktgrootte
beperk, bly mortaliteit en morbiditeit steeds hoog. Isgemiese prekondisionering
(IPK) beskik oor die potensiaal om miokariale isgemie/herperfusie skade te
verminder deur beskermende seinoordragpaaie tydens miokardiale herperfusie te
aktiveer en sodoende die pasiënte wat aan koronêre arterie siekte ly, se
prognose te verbeter.
Isgemiese prekondisionering verwys na die verskynsel waartydens kort episodes
van isgemie opgevolg deur herperfusie, die miokardium teen ‘n daaropvolgende
langdurige isgemiese insident beskerm. Hierdie beskerming word gereflekteer in
die beperking van infarktgrootte en verbeterde funksionele herstel van die
isgemiese hart tydens herperfusie. Ten spyte van intensiewe navorsingspogings
is die presiese meganisme van endogene beskerming tydens IPK nog nie ten
volle ontrafel nie. Die identifisering van die miokardiale herperfusie fase se
kritiese “vensterperiode” van beskerming, noodsaak ‘n volledige analise van die
seinoordragpaaie wat geaktiveer word tydens die herperfusie fase na IPK.
In die lig van bogenoemde, was die doel van die huidige studie om die volgende
te ondersoek:
i. die betrokkenheid van die RISK seinoordragpad en p38 MAP kinase
tydens vroeë en laat herperfusie na IPK
ii. die betrokkenheid van “heat shock protein-27” (HSP-27), “heat shock
protein- 70” (HSP-70), GSK -3β, CAMKII, AMPK en die transkripsie faktor,
CREB, in die konteks van IPK tydens vroeë herperfusie
iii. die betrokkenheid van outofagie en apoptose tydens vroeë en laat
herperfusie na IPK
iv. die korrelasie van die proteïenkinases met die hemodinamiese parameters
van die hart v. die meganisme van IPK deur middel van twee dimensionele proteomika
Metodes: Die geïsoleerde werkende rothart model, met funksionele herstel as
eindpunt, is gebruik. Harte is geprekondisioneer (IPK) met 3x5 min globale
isgemie, afgewissel met 5 min herperfusie. Daarna is harte blootgestel aan 25
min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie,
waartydens harte gevriesklamp is. Alternatiewelik, is harte blootgestel aan 30 min
herperfusie ten einde funksionele herstel te meet en hemodinamiese parameters
te registreer. Nie-geprekondisioneerde (Non-IPK) harte is gestabiliseer vir 30
min, waarna dit onderwerp is aan 25 min volgehoue globale isgemie, gevolg deur
5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is vir westelike
klad analise. Alternatiewelik, is Non-IPK harte onderwerp aan 30 min herperfusie
om te dien as kontrole vir die 30 min IPK groep. Aktivering van die
proteïenkinases is bepaal deur westelike klad analise.
Vir die proteomiese studie, is onderskeidelik mitokondriale en sitosoliese
proteïene geïsoleer en geskei in die eerste dimensie met behulp van isoelektriese
fokusering, gevolg deur skeiding in die tweede dimensie met behulp
van twee dimensionele gel elektroforese. Die PDQuest sagteware program is
gebruik om proteïenkolle te identifiseer wat statisties beduidende verskille toon.
Proteïenkolle van belang is uitgesny en onderwerp aan in-gel tripsinering en die
peptiede wat sodoende verkry is, is deur middel van massa spektrometrie
geanaliseer. Proteïene is geïdentifiseer deur Mascot en die Swiss Prot databasis. Resultate: Westelike klad analise het aangetoon dat die RISK pad en p38 MAPK
geaktiveer is tydens vroeë herperfusie, maar die aktivering word nie volgehou
tydens die hele herperfusie periode nie. Outofagie word gestimuleer tydens die
vroeë herperfusie fase; dit word onderdruk in die middel herperfusie fase en
bereik ‘n tweede piek van stimulering in die laat herperfusie fase. Die
proteomiese analise het onderskeidelik twintig differensieel gereguleerde
mitokondriale proteïene en ses en dertig differensieel gereguleerde sitosoliese
proteïene geïdentifiseer tussen Non-IPK en IPK. Die grootste persentasie van
hierdie proteïene is direk betrokke by miokardiale energie metabolisme. CAMKII
is geidentifiseer as ‘n unieke merker van funksionele herstel in IPK tydens
reperfusie. Gevolgtrekking: Aktivering van die meeste van die proteïenkinases wat
ondersoek is in die huidige studie, is geassosieer met die hemodinamiese
parameters van die hart, in plaas van funksionele herstel. Die resultate het
aangetoon dat die varierende patrone van kinase aktivering toegeskryf kan word
word aan verskille in harttempo en die effek daarvan (ejeksie fraksie, minimum
en maksimum tempo van kontraksie), as gevolg van simpatiese stimulasie
toegeskryf aan sielkundige stres in die diere voor slagting. Proteomiese analise
het getoon dat IPK harte wat faal na isgemie/reperfusie metabolies
gekompromiseer is en “slegter daaraan toe” is, in vergelyking met Non-IPK harte.
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The effects of chronic melatonin treatment on myocardial function and ischaemia and reperfusion injury in a rat model of diet-induced obesityNduhirabandi, Frederic 03 1900 (has links)
Thesis (MScMedSc)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Obesity is a major risk factor for ischaemic heart disease. Obesity-induced metabolic
abnormalities have been associated with increased oxidative stress which may play an
important role in the increased susceptibility to myocardial dysfunction and ischaemiareperfusion
(I/R) injury seen in obesity. The pineal gland hormone, melatonin, has powerful
antioxidant properties. Previous studies have shown that short-term or acute melatonin
administration protects the normal healthy heart of lean animals against I/R damage. However,
the effects of melatonin on the heart in obesity remain unknown. Moreover, the myocardial
signalling mechanisms associated with the cardioprotective effects of melatonin have not been established. Using a rat model of diet induced obesity, we set out to: 1) investigate the effects of chronic
melatonin administration on the development of diet-induced systemic alterations including
biometric and metabolic parameters and oxidative stress, 2) determine whether chronic
melatonin treatment protects the myocardium against ischaemia-reperfusion injury, and 3)
determine whether melatonin treatment confers cardioprotection by altering the reperfusion
injury salvage kinase (RISK) pathway signalling and the pro-apoptotic p38 MAPK, AMPK and
GLUT-4 expression. Male rats weighing 200±20g were randomly allocated to four groups: 1) C, control rats
receiving a standard commercial rat chow and drinking water without melatonin; 2) CM, control
rats receiving melatonin (4mg/kg/day) in drinking water; 3) D, diet-induced obesity rats,
receiving a high calorie diet and drinking water without melatonin; 4) DM, diet-induced obesity
rats, receiving melatonin in drinking water. After 16 weeks of treatment and feeding, rats were
weighed and blood and myocardial tissue collected to document biochemical and molecular
biological changes. Hearts were perfused on the isolated working rat heart perfusion apparatus
for the evaluation of myocardial function and infarct size. The Reperfusion Injury Salvage
Kinases (RISK) pathway (PKB/Akt (Ser-473), ERK p42/ p44) and p38 MAPK (mitogenactivated
protein kinase) were investigated in pre-and post-ischaemic hearts using Western
blotting techniques. Post-ischaemic activation of AMPK (5’AMP-activated protein kinase) (Thr-
172) and GLUT-4 (glucose transporter) expression were also investigated. Serum and
baseline myocardial glutathione (GSH) content were measured. In addition, serum lipid peroxidation products: thiobarbituric reactive substances (TBARS), conjugated dienes (CD)
and lipid hydroperoxide (LOOH), were also determined. The high-calorie diet caused increases in body weight, visceral adiposity, heart weight, serum
insulin, leptin, blood triglycerides, and low HDL-cholesterol levels. Blood glucose levels were
similar for both diet fed rats and controls. Myocardial glutathione, serum glutathione, total
cholesterol, TBARS, LOOH, CD as well as total cholesterol (TC) levels were not affected by
the high calorie diet. Chronic melatonin treatment reduced body weight gain, visceral
adiposity, heart weight, blood triglycerides, serum insulin, HOMA index, serum leptin (DM vs D,
p<0.01), and increased blood HDL-C in diet treated rats while there was no effect on these
parameters in control rats, despite the reduction in body weight, heart weight and visceral
adiposity. Melatonin treatment had no effect on myocardial or serum GSH and LOOH in either
control or diet animals. It however reduced TBARS and CD in the diet and control groups,
respectively. At baseline, chronic melatonin treatment caused a significant increase in
phospho-PKB/total PKB ratio and a concomitant reduction in phospho-p38 MAPK/total p38
MAPK ratio of control hearts while there were no such effects on diet-induced-obesity hearts.
Infarct size was significantly reduced by melatonin in both diet and control groups (DM:
16.6±2.0%; D: 38.4±2.6% (p < 0.001), and CM: 12.8±1.5%; C: 30.4±1.0%, p<0.001). After
coronary artery occlusion and 30 minutes of reperfusion, melatonin increased percentage
recovery of aortic output (DM: 28.5±6.5%; D: 6.2±6.2%, p<0.01), cardiac output (DM:
44.4±5.2%; D: 26.6±5.1%, p < 0.01) and total work (DM: 34.5±5.6%; D: 20.4±7.9%, p<0.05) of
diet-induced obesity hearts, while having no effect on control hearts. During reperfusion, hearts
from melatonin treated rats had increased activation of PKB/Akt (p<0.01), ERK42/44 (p<0.05),
and reduced p38 MAPK activation (p<0.05). There was no difference in post-ischaemic
activation of AMPK (Thr-172) and GLUT-4 expression in either control or diet fed rats. We successfully demonstrated that chronic melatonin treatment prevented the development of
diet-induced metabolic abnormalities and improved ex vivo myocardial function. Melatonin
protected the heart against ischaemia-reperfusion injury that was exacerbated in obesity. This
was achieved by activation of the RISK pathway. The antioxidant properties of melatonin were
involved in these cardioprotective effects. / AFRIKAANSE OPSOMMING: Vetsug of obesiteit is een van die hoof risikofaktore vir iskemiese hartsiekte. Obesiteitgeinduseerde
metaboliese abnormaliteite gaan met verhoogde oksidatiewe stres gepaard wat
op sy beurt ‘n belangrike rol mag speel in die miokardiale wanfunksie en verhoogde
vatbaarheid vir iskemie-herperfusie (I/H) beskadiging, kenmerkend van vetsug. Melatonien, die
hormoon afgeskei deur die pineaalklier, is ‘n kragtige anti-oksidant. Vorige studies het getoon
dat kort-termyn of akute toediening van melatonien die normale hart van gesonde diere teen
I/H beskadiging deur middel van sy anti-oksidant aksies beskerm. Die effek van melatonien op
die hart in obesiteit is egter nog onbekend. Hierbenewens is die miokardiale seintransduksie
meganismes geassosieer met die beskermende effekte van die hormoon nog nie ontrafel nie. ‘n Model van dieet-geinduseerde obesiteit in rotte is gebruik om die volgende te bepaal: (i) die
effek van kroniese melatonientoediening op die ontwikkeling van dieet-geinduseerde
sistemiese veranderinge soos biometriese en metaboliese parameters en oksidatiewe stres (ii)
die effek van kroniese melatonienbehandeling op die respons van die hart op I/H beskadiging
en (iii) die rol van herperfusie beskadiging op die aktivering van PKB/Akt en ERK42/44 (die sg
RISK seintransduksiepad), die pro-apoptotiese p38MAPK, AMPK sowel as die uitdrukking van
GLUT-4. Manlike Wistar rotte (200±20g) is ewekansig in vier groepe verdeel: (i) C, kontrole rotte wat ‘n
standaard rotdieet en drinkwater sonder melatonien ontvang (ii) CM, kontrole rotte wat
melatonien (4mg/kg/dag) ontvang (iii) D, dieet-geϊnduseerde vet rotte wat ‘n hoë kalorie dieet
en drinkwater sonder melatonien ontvang (iv) DM, dieet-geϊnduseerde vet rotte wat melatonien
(4mg/kg/dag) in die drinkwater ontvang. Na 16 weke van behandeling, is die rotte geweeg,
bloed en hartweefsel gekollekteer vir biochemiese en molekulêre biologie bepalings. Harte is
geperfuseer volgens die werkhartmodel, blootgestel aan iskemie/herperfusie vir evaluering van
funksionele herstel en infarktgrootte. Uitdrukking en aktivering van PKB/Akt (Ser-473),
ERKp42/p44 en p38MAPK van pre-en postiskemiese hartweefsel is met behulp van Western
blot bepaal. Postiskemiese aktivering van AMPK (5’AMP-aktiveerde proteϊen kinase) (Thr-172)
en GLUT-4 (glukose transporter) is op soortgelyke wyse bepaal. Serum en basislyn
hartweefsel glutatioon (GSH) inhoud asook tiobarbituursuur reaktiewe substans (TBARS),
gekonjugeerde diene (CD) en lipiedhidroperoksied (LOOH) konsentrasies is bepaal. Resultate
Die hoë kalorie diet het ‘n toename in liggaamsgewig, visserale vet, hartgewig, serum insulien,
leptien, plasma trigliseried en lae HDL-cholesterol vlakke teweegebring. Bloed glukosevlakke
was egter dieselfde in die vet en kontrole rotte. Miokardiale glutatioon, serum glutatioon, totale
cholesterol, TBARS, LOOH, CD is nie deur die dieet beinvloed nie. Chroniese melatonien
behandeling het die liggaamsgewig, visserale vet, hartgewig, plasma trigliseried, serum
insulien en leptien, HOMA indeks verlaag (DM vs D, p<0.05) en die HDL-cholesterol verhoog
in die dieetrotte, terwyl dit geen effek op hierdie parameters in kontrole rotte gehad het nie
(uitgesonderd ‘n afname in liggaamsgewig, hartgewig en visserale vet). Melatonien
behandeling het geen effek op hart of serum GSH en LOOH in kontrole en vet rotte gehad nie.
Dit het egter die TBARS en CD in beide vet en kontrole rotte verlaag. Chroniese melatonien
toediening het ‘n beduidende toename in basislyn fosfo PKB//totale PKB ratio en ‘n afname in
fosfo p38MAPK/totale p38MAPK ratio teweegebring in harte van kontrole rotte, maar
soortgelyke effekte is nie in die harte van die vet rotte waargeneem nie. Infarktgrootte is
beduidend deur melatonienbehandeling verlaag in beide dieet en kontrole groepe (DM: 16.6±
5.2%, D: 38.4 ±2.6% (p<0.001); CM: 12.8± 1.5%; C 30.4±1.0 (p<0.001). Na koronere arterie
afbinding en 30 min van herperfusie, het melatonien die persentasie herstel van aorta omset
(DM: 28.5± 6.5%; D: 6.2± 6.2%, p<0.01), kardiale omset ( DM: 44.4± 5.2%D: 26.6±5.1%,
p<0.01) en totale werk (DM: 34.5 5.6%; D 20.4± 7.9%, p<0.05) in die harte van dieetrotte
verbeter, terwyl dit sonder effek was in kontrole harte. Tydens herperfusie het harte van
melatonienbehandelde rotte verhoogde aktivering van PKB/Akt (p<0.01) en ERKp42/p44
(p<0.05) getoon, terwyl aktivering van p38MAPK verlaag is (p<0.05). Geen verskil in
postiskemiese aktivering van AMPK en GLUT-4 uitdrukking is in beide kontrole en dieetrotte
waargeneem nie. Ons het daarin geslaag om aan te toon dat chroniese melatonienbehandeling die ontwikkeling
van dieet-geϊnduseerde metaboliese abnormaliteite beduidend kan voorkom en ex vivo
miokardiale funksie verbeter. Melatonien het ook die hart teen iskemie/herperfusie beskadiging
beskerm in beide kontrole en dieetrotte. Bogenoemde veranderinge het met aktivering van
PKB/Akt en ERKp42/p44 gepaard gegaan. Die anti-oksidant effekte van melatonien was
heelwaarskynlik hierby betrokke.
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Possible mechanisms for levosimendaninduced cardioprotectionGenis, Amanda 12 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / Background and purpose. To limit ischaemic injury, rapid restoration of coronary
blood flow is required, which will in turn reduce infarct size. However, reperfusion
itself causes myocyte death – a phenomenon termed lethal reperfusion-induced
injury, which limits protection of the ischaemic myocardium. Thus the reperfusion
of irreversibly damaged myocytes may accelerate the process of cell necrosis.
Additive protection of the ischaemic myocardium in the form of adjunct therapy
remains a topic of intensive research. Levosimendan, a calcium sensitizing agent
with positive inotropic effects has in several studies been found to alleviate the
damaging effects of reperfusion injury. Levosimendan has been shown to be a
KATP channel opener. These channels have been implicated to play an important
role in ischaemic preconditioning (IPC). With this knowledge, the aim of this study
was to determine whether levosimendan and IPC have certain cardioprotective
mechanisms in common and whether protection with pharmacological
preconditioning could be elicited with levosimendan. In this study, we investigated
whether: 1) the isolated guinea pig heart could be protected by ischaemic
preconditioning (IPC) and postconditioning (IPostC), 2) the heart could be
pharmacologically pre- and postconditioned, using levosimendan (LPC & LPostC),
3) a combination of IPC & LPC had an additive protective effect on the heart, 4)
the KATP (both mitochondrial and sarcolemmal) channels are involved in this
protection and 5) the pro-survival kinases of the RISK (reperfusion injury salvage
kinase) pathway are involved.
Experimental approach. Isolated perfused guinea pig hearts were subjected to
three different IPC protocols (1x5, 2x5 and 3x5 minutes of ischaemia) or
levosimendan (0.1μM) preconditioning, before coronary artery occlusion (CAO –
40min@36.5ºC), followed by 30 minutes of reperfusion. Hearts were also
subjected to a combination of IPC & LPC, to establish whether they had additive
protective effects. In addition, hearts were pre-treated with levosimendan directly
before induction of sustained ischaemia (without washout of the drug –
levosimendan pre-treatment (LPT)) for 10min. With the postconditioning protocol,
iii
the hearts were subjected to 3x30second cycles of ischaemia/reperfusion or
levosimendan/vehicle. In a separate series of experiments, hearts were treated
with KATP channel blockers (for both sarcolemmal & mitochondrial), before LPC,
LPT and LPostC. The endpoints that were measured were: cardiac reperfusion
function, myocardial infarct size and RISK pathway expression and
phosphorylation (PKB/Akt and extracellular signal-regulated kinase – ERK42/44).
Results. IPC, IPostC, LPC & LPostC decreased myocardial infarct size
significantly compared with their controls (21.9±2.2%, 21.4±2.2%, 20.6±3.1% and
20.6±1.8% respectively vs. 46.4±1.8% for controls, p<0.05). The combination of
IPC & LPC had no additive protective effect. Pre-treating the hearts with
levosimendan (without washout), before index ischaemia, proved to be the most
effective method of cardioprotection (infarct size: 5.8±0.9% vs. 46.4±1.8% for
controls, p<0.001). With LPT a significant increase (p < 0.05 vs. control) in
phosphorylation of ER42/44 was also observed. An increase in the activity of one
of the RISK pathway kinases, ERK42/44 seems to be one of the reasons for LPT’s
efficacy. Treating the hearts with KATP channel blockers before subjecting them to
LPC, LPT & LPostC abolished the protective effects induced by levosimendan,
suggesting a role for the sarcolemmal and mitochondrial KATP channels in
levosimendan-induced cardioprotection.
Conclusions and implications. 1) Isolated guinea pig hearts could be pre- and
postconditioned within the setting of ischaemia, 2) Hearts could be
pharmacologically pre- and postconditioned with levosimendan, 3) levosimendan
pre-treatment is the most effective way to reduce infarct size, possibly acting by
increasing the phosphorylation of ERK42/44, 4) Myocardial protection was not
increased by combining IPC & LPC (suggesting similar mechanisms of protection),
5) LPC, LPT and LPostC were abolished by both sarcolemmal and mitochondrial
KATP channel blockers.
.LPC and especially LPT, could be useful before elective cardiac surgery while
LPostC may be considered after acute coronary artery events.
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The effect of hypoxia on nitric oxide and endothelial nitric oxide synthase in the whole heart and isolated cardiac cells: the role of the PI3–K / PKB pathway as a possible mediator.Chamane, Nontuthuko Zoleka Lynette 03 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / In the heart, endothelial nitric oxide synthase (eNOS) is regarded as the most
important constitutively expressed enzymatic source of nitric oxide (NO), a
major cardiac signalling molecule. On the whole, NO is regarded as a
cardioprotective molecule. The role of eNOS during ischaemia / hypoxia is
controversial; however, it is generally accepted that ischaemia / hypoxia
results in increased cardiac NO production. Most studies focus either on the
whole heart or isolated cell models. As yet, no study has compared findings
with regard to NO metabolism in these two distinct models, in a single study.
We hypothesise that observations in a whole heart model with regard to
increased NO production and eNOS involvement in ischaemia are the result
of events on cellular level and that the increase in NO production observed
during hypoxia in cardiomyocytes and endothelial cells is at least in part due
to the increase in expression and / or activation of eNOS. Furthermore, we
hypothesize that these effects are mediated via the PI3-K / PKB pathway. We
aimed to measure and compare NO-production and eNOS expression and
activation in the whole heart and isolated cardiac cells and measure PKB
expression and activation in the cells under normoxic and ischaemic / hypoxic
conditions. We also aimed to determine the effects of PI3-K / PKB pathway
inhibition on NO production and eNOS expression and activation in isolated
cardiac cells under normoxic and hypoxic conditions. Adult rat hearts were
perfused and global ischaemia induced for 15 and 20 min. Tissue
homogenates of perfused hearts were used for the measurement of nitrites
and determination of expression and activation of eNOS. Expression of eNOS
in the heart was also determined by immunohistochemical (IHC) analysis.
Cardiomyocytes were isolated from adult rat hearts by collagenase-perfusion,
and adult rat cardiac microvascular endothelial cells (CMEC) purchased
commercially. In the cells, hypoxia was induced by covering cell pellets with
mineral oil for 60 min. Cell viability was determined by trypan blue and
propidium iodide (PI) staining and intracellular NO production measured by
FACS analysis of the NO-specific probe, DAF-2/DA and by measurement of
nitrite levels (Griess reagent). Results show that in ischaemic hearts, nitrite
production increased by 12 % after 15 min ischaemia and 7 % after 20 min
ischaemia. Total eNOS expression remained unchanged (Western Blot and
IHC) and activated eNOS (phospho-eNOS Ser1177) increased by 38 % after 15
min ischaemia and decreased by 43% after 20 min ischaemia. In the cells,
both viability techniques verified that the hypoxia-protocol induced significant
damage. In isolated cardiomyocytes, NO-production increased 1.2-fold (by
DAF-2/DA fluorescence), total eNOS expression increased 2-fold and
activated eNOS increased 1.8-fold over control. In CMECs, NO-production
increased 1.6-fold (by DAF-2/DA fluorescence), total eNOS increased by 1.8-
fold and activated eNOS by 3-fold. With regards to our PI3-K / PKB
investigations, results showed an increase of 84 % and 88 % in expression
vii
and activation of PKB (phospho Ser473) in hypoxic cardiomyocytes,
respectively. In hypoxic CMECs, there was no change in PKB expression but
there was a 69 % increase in phosphorylated PKB. NO production in
wortmannin-treated hypoxic cardiomyocytes decreased by 12 % as compared
to untreated hypoxic cells. In treated hypoxic CMECs, NO production
decreased by 58 % as compared to untreated hypoxic cells. Treatment with
wortmannin did not change the expression of eNOS protein in the
cardiomyocytes, however, activated eNOS decreased by 41 % and 23 %
under baseline and hypoxic conditions in treated cells respectively. There was
a significant increase in NO production after exposure to O2 deficient
conditions in all models investigated, a trend similar to what previous studies
in literature found. However, the source of this NO is not fully understood
although it has been discovered that NOS plays a role. Our data reveals
similar trends in 15 min ischaemia in whole hearts and 60 min hypoxia in the
cells; however, the trends observed at 20 min ischaemia are in conflict with
our cell data (i.e. decrease in activated eNOS). This may be due to the
severity of the ischaemic insult in whole hearts and/or the presence of other
cell types and paracrine factors in the whole heart. Hypoxia increased the
activation of PKB in the isolated cardiac cells. Inhibition of the PI3-K / PKB
pathway reduced NO production and hypoxia-induced eNOS activation in
cardiomyocytes. In conclusion, we have, for the first time, demonstrated that
the increase in NO production during hypoxia is due (at least in part) to an
increase in eNOS phosphorylation at Ser1177 and that this is mediated via the
PI3-K / PKB pathway.
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