Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: It is well-established that transient activation of the β-adrenergic signalling pathway with ligands such as isoproterenol, formoterol and dobutamine, elicits cardioprotection against subsequent long periods of ischaemia. Initially the focus was on the β1- and β2-adrenergic receptors (β1-AR, β2-AR), but recently the β3-AR also emerged as a potential target in the treatment of heart disease. In heart failure, β1- and β2-AR are typically known to be down-regulated while β3-ARs, on the other hand, are up-regulated (Moniotte et al., 2001). Thus, it has become important to examine the significance of the β3-AR and its downstream signalling under similar states of stress. It has been shown that β3-AR stimulation is resistant to short term agonist-promoted desensitization in vitro and in vivo (Liggett et al., 1993) and after being activated, this receptor is able to convey continual intracellular signals (Lafontan et al., 1994). Thus, it could be an ideal target for therapeutic intervention, also in ischaemic heart disease. We hypothesized that selective β3-AR stimulation during ischaemia / reperfusion may be cardioprotective, whereas selective inhibition of this receptor may prove useful in the end stages of sustained ischaemia and early reperfusion. Methods: The isolated working rat heart, subjected to 35 min of regional ischaemia (RI) and 60 min reperfusion was used as model. The β3-AR agonist (BRL37344) (1 μM) or antagonist (SR59230A) (0.1 μM) were applied as follows: (i) before 35 min RI (PT), (ii) during the last 10 min of RI (PerT) and /or (iii) at the onset of reperfusion (PostT) and (iv) administration of BRL37344 during the last 10 min of RI BRL37344 (PerT) was followed by SR59230A during first 10 min of reperfusion SR59230A (Post). The contribution of nitric oxide synthase (NOS) in β3-AR was assessed, using the non-specific NOS inhibitor, L-NAME (50 μM). Endpoints were functional recovery and infarct size. In another set of experiments BRL37344 and SR59230A were applied according to the same protocols, but the left ventricle was dissected from the heart and freeze clamped at 10 min reperfusion for Western blot analysis of extracellular signal-regulated kinase (ERK p44/p42), protein kinase B (PKB/Akt), glycogen synthase kinase-3β (GSK-3β), and endothelial nitric oxide synthase (eNOS). Data were analyzed with one or two-way analysis of variance (ANOVA). Results: Administration of the selective β3-AR agonist (BRL37344) (1μM) before 35 min RI (BRL37344 (PT), significantly reduced infarct size when compared to the non-pretreatment group (NPT) (21.43±2.52 vs 43.17±1.20, p < 0.001). BRL37344 had similar effects on infarct size when applied during the last 10 min of regional ischaemia BRL37344 (PerT) (14.94±2.34, vs NPT, p < 0.001) or at the onset of reperfusion BRL37344 (PostT) (19.06±1.81, vs NPT, p < 0.001). When BRL37344 was applied as a (PerT+PostT) strategy, infarct size was once again significantly reduced (20.55±2.01 vs 43.17±1.20, p <0.001). In contrast, administration of the β3-antagonist SR59230A according to the same protocol did not reduce infarct size and values similar to those of untreated hearts (NPT) were obtained. Surprisingly, when BRL37344 was applied during the last 10 min of regional ischaemia followed by the administration of the β3-AR antagonist (SR59230A) at the onset of reperfusion, [BRL37344 (PerT) & SR59230A (PostT)], infarct size was significantly reduced to 20.78±3.02 (p <0.001 vs NPT and SR59230A (PerT + PostT). Involvement of nitric oxide (NO) was shown since the reduction in infarct size elicited by BRL37344 was totally abolished by, L-NAME, when administered in combination with BRL37344 for 10 minutes prior to RI or at the onset of reperfusion for 10 minutes (% infarct size: 41.48±3.18 and 35.75±3.54, p <0.001 vs BRL37344 (PT) and BRL37344 (PostT), respectively. Western blot results show that PKB/Akt is activated by BRL37344 regardless of the time of administration. The intervention BRL37344 (PerT+PostT), exhibited the most significant phosphorylation of PKB/Akt (fold increase: 14.2±3.71, p<0.01 vs NPT and p<0.05 vs BRL37344 (PostT). In addition, BRL37344 (PT), (PerT), (PostT) and [BRL37344 (PerT) +SR59230A (PostT)] showed significant activation of this kinase (2.92±0.22, 5.54±0.43, 4.73±0.47, and 6.60±0.78, respectively). ERKp44/p42 however, was not significantly activated by any of the treatments. Phosphorylation of eNOS and GSK-3β was significant only in the BRL37344 (PerT+PostT) and [BRL37344 (PerT) + SR59230A (PostT)] groups. The activation of eNOS-S-1177 in the BRL37344 (PerT+PostT) group was (2.82±0.46, p<0.01 and 0.05 vs NPT and BRL37344 (PostT), respectively) and in the [BRL37344 (PerT) + SR59230A (PostT)] group was (2.26±0.48, p<0.05 vs NPT). A very significant increased phosphorylation of GSK-3β was seen in the same two groups (68.8±7.73, p<0.001 vs NPT and 25.5±5.42 vs NPT, p<0.05, respectively). Conclusion: β3-AR has potent cardioprotective effects when administered either before, during and after ischaemia during early reperfusion as indicated by the reduction in infarct size as well as activation of PKB, GSK-3β and eNOS. These beneficial effects can be linked to NO production through activation of eNOS. / AFRIKAANSE OPSOMMING: Dit is bekend dat verbygaande aktivering van die β-adrenerge seinpad, met ligande soos isoproterenol, formoterol en dobutamien, die hart teen daaropvolgende lang periodes van iskemie beskerm. Aanvanklik was die fokus op die β1- en β2-adrenerge reseptore (β1-AR, β2-AR); maar onlangs is ook die β3-AR as 'n potensiële teiken in die behandeling van hartsiektes ge-eien. In hartversaking, is dit bekend dat β1- en β2-AR afreguleer word, terwyl β3-ARs, aan die ander kant, opreguleer word (Moniotte et al., 2001). Dit het dus belangrik geword om die belang van die β3-AR en sy stroomaf seinpad onder soortgelyke strestoestande te ondersoek. Dit is bewys dat β3-AR stimulasie teen korttermyn agonis geïnduseerde desensitisering in vitro en in vivo bestand is (Liggett et al., 1993) en wanneer geaktiveer, is hierdie reseptor in staat om intrasellulêre seine voortdurend oor te dra (Granneman, 1995). Dit kan dus ‘n ideale teiken vir terapeutiese intervensie wees, ook in iskemiese hartsiekte. Ons hipotetiseer dat selektiewe β3-AR stimulasie tydens iskemie / reperfusie kardiobeskermende mag wees, terwyl selektiewe inhibisie van hierdie reseptor effektief kan wees in die eindstadia van volgehoue iskemie en vroeë herperfusie. Metodes: Die geïsoleerde werkende rothart, onderwerp aan 35 min van streeksiskemie (SI) en 60 min herperfusie, is as model gebruik. Die β3-AR agonis (BRL37344) (1μM) of antagonis (SR59230A) (0.1 μM), is as volg toegedien: (i) voor 35 min SI (PT), (ii) gedurende die laaste 10 min van SI (PerT) en / of (iii) tydens die aanvang van herperfusie (PostT) en (iv) gedurende die laaste 10 min van SI is BRL toediening BRL37344 (PerT) gevolg deur SR59230A tydens die eerste 10 min van herperfusie SR59230A (Post). Die rol van stikstofoksiedsintase (NOS) in β3-AR is met behulp van die nie-spesifieke NOS inhibitor, L-NAME (50 μM) ondersoek. Eindpunte was funksionele herstel tydens herperfusie en infarktgrootte. In 'n ander reeks eksperimente is BRL37344 en SR59230A volgens dieselfde protokolle toegedien, maar die linker ventrikel is uit die hart gedissekteer na 10 min herperfusie en gevriesklamp vir Western klad analise van ekstrasellulêre-sein gereguleerde kinase (ERK p44/p42), proteïen kinase B (PKB/Akt), glikogeen sintase kinase-3β (GSK-3β), en endoteel stikstofoksied- sintase (eNOS). Data is met een of twee-rigting variansie analise (ANOVA) ontleed. Resultate: Administrasie van die selektiewe β3-AR agonis (BRL37344) (1μM) voor 35 min SI BRL37344 (PT), het die infarktgrootte beduidend verminder vergeleke met die nie-behandelde groep (NPT) (21.43±2.52 vs 43.17±1.20, p<0.001). BRL37344 het ‘n soortgelyke effek op infarktgrootte wanneer dit gedurende die laaste 10 min van streeksiskemie BRL37344 (PerT) (14.94±2.34, vs NPT, p<0.001) of by die aanvang van herperfusie (BRL37344 (PostT) (19.06±1.81, vs NPT, p<0.001) toegedien word. Wanneer BRL37344 as 'n (PerT+PostT) strategie toegedien is, was infarktgrootte weereens beduidend verlaag (20.55±2.01 vs 43.17±1.20, p<0.001). In teenstelling hiermee, het administrasie van die β3-antagonis SR59230A volgens dieselfde protokol, nie infarktgrootte verminder nie en waardes soortgelyk aan dié van onbehandelde harte (NPT) is verkry. Interessant, wanneer BRL37344 gedurende die laaste 10 min van streeksiskemie toegedien is, gevolg deur die administrasie van die β3-AR antagonis (SR59230A) by die aanvang van herperfusie, [BRL37344(PerT) & SR59230A(PostT)], was infarktgrootte aansienlik verminder tot 20.78±3.02 (p<0.001 vs NPT en SR59230A (PerT+PostT). Die betrokkenheid van stikstofoksied (NO) is waargeneem deurdat die vermindering in infarktgrootte ontlok deur BRL37344, heeltemal deur L-NAME opgehef is, wanneer dit in kombinasie met BRL37344 vir 10 minute voor SI of by die aanvang van herperfusie vir 10 minute toegedien is (% infarktgrootte: 41.48±3.18 en 35.75±3.54, p<0.001 vs BRL37344 (PT) en BRL37344 (PostT) onderskeidelik). Western kladresultate toon dat PKB/Akt deur BRL37344 geaktiveer word ongeag die tyd van die administrasie. Die intervensie BRL37344 (PerT+PostT), toon die mees beduidende fosforilering van PKB/Akt (voudige toename: 14.2±3.71, p<0.01 vs NPT en p<0.05 vs BRL37344 (PostT). Daarbenewens het BRL37344 (PT), (PerT), (PostT) en [BRL37344 (PerT) + SR59230A (PostT)] ook beduidende aktivering van hierdie kinase tot gevolg gehad (2.92±0.22, 5.54±0.43, 4.73±0.47 en 6.60±0.78, onderskeidelik). ERKp44/p42 is egter nie deur enige van die behandelings geaktiveer nie. Fosforilering van eNOS en GSK-3β was net beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. Die aktivering van eNOS-S-1177 was beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. 'n Baie beduidende toename in fosforilering van GSK-3β is in dieselfde twee groepe (68.8±7.73, p<0.001 en 25.5±5.42, p<0.05 vs NPT onderskeidelik) waargeneem. Gevolgtrekking: β3-AR het kragtige kardiobeskermende effekte wanneer dit, hetsy voor, tydens en na iskemie gedurende vroeë herperfusie toegedien word, soos deur die vermindering in infarktgrootte sowel as die aktivering van PKB, GSK-3β en eNOS aangedui is. Hierdie voordelige effekte kan aan NO produksie deur aktivering van eNOS gekoppel word.

The Effect of Sulindac was studied on an animal model of ischemic stroke. Sulindac, a non steroid anti inflammatory drug (NSAID) could protect cell death due to hypoxia/reoxygenation. This drug was given 2 days before and 24 hrs after ischemia until animals were sacrificed on 3rd or 11th day. Infarct size was measured for these animals. Sulindac induced Hsp 27 in ischemic penumbra and core on Day 3 & 11 with uncoated nylon suture which shows its cell-survival and anti-apoptotic activity. Also, it increased expression of cell survival markers such as Akt, Bcl2 & Grp 78 in ischemic penumbra and core. With silicon suture it reduced expression of Hsp 27 in ischemic penumbra and core, alleviating cell stress and having pro-survival and anti-stress effects. In conclusion sulindac may have excellent potential as neuro protective agent against oxidative stress in cerebral ischemia. / by JIgar Modi. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Stroke is the third leading cause of mortality in the United States, and so far, no clinical interventions have been proved truly effective in stroke treatment. Stroke my result in hypoxia, glutamate release and oxidative stress, etc. The purpose of this dissertation study is to evaluate the neuroprotective effects of four drugs (taurine, G-CSF sulindac and DETC-MeSO) on PC12 cell line or primary cortical neuronal cell culture, and to understand the protective mechanisms underlying in three stroke-related models : hypoxia, excessive glutamtate and oxidative stress. In the first part of this dissertation, we studied the neuroprotection of taurine against oxidative stress induced by H2O2 in PC12 cells. Our results show that extracellular taurine exerts a neuroprotective function by restoring the expression of Bcl-2 and downregulation of the three Endoplasmic Reticulum (ER) stress markers : GRP78, Bim and CHOP/GADD153, suggesting that ER stress can be provoked by oxidative stress and can be suppressed by taurine. In the second part, glutamate excitotoxicity-induced ER stress was studied with dose and time as variables in primary cortical neurons. The results demonstrate that glutamate excitotoxicity leads to the activation of three ER stress pathways (PERK, ATF6 and IRE1) by initiating PERK first, ATF6 second and IRE1 pathway last. The third part of this dissertation studied the robust and beneficial protection of taurine in cortical neurons under hypoxia/reoxygenation or glutamate toxicity condition. We found that taurine suppresses the up-regulation of GRP778, Bim, caspase-12 and GADD153/CHOP induced by excessive glutamate or hypoxia/reoxygenation, suggesting that taurine may exert a protective function against hypoxia/regeneration by reducing the ER stress. / Moreover, taurine can down-regulate the ratio of cleaved ATF6 and full length ATF6, and p-IRE1 expresssion, indicating that taurine inhibits the ER stress induced by hypoxia/reoxygenation or glutamate through suppressing ATF6 and IRE1 pathways. In the fourth part, the synergistic benefits of the combination of taurine and G-CSF, and the neuroprotective effects of G-CSF, sulindac or DETC-MeSO are studied in cortical neurons. Our results show that G-CSF, sulindac or DETC-MeSO can highly increase the neuron visibility by inhibiting ER stress induced by hypoxia/reoxygenation or glutamate toxicity. Furthermore, we proved that G-CSF or sulindac can significantly inhibit the activation of ATF6 or IRE1 pathway stimulated by hypoxia/reoxygenation, and DETC-MeSO can suppress the activation of both PERK and IRE1 pathways in primary neuron cultures. These findings provide promising and rational strategies for stroke therapy. / by Chunliu Pan. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Background. Carotid stenosis is an important cause of stroke. Carotid endarterectomy is a means of reducing the burden of stroke but is of marginal benefit in individuals with asymptomatic carotid stenosis. The identification of factors associated with increased risk of cerebral ischemic events would help select individuals who may obtain a greater benefit. / Methods. A comprehensive search was performed to identify studies examining risk factors for cerebral ischemic events in patients with asymptomatic carotid stenosis. Inclusion criteria were defined a priori. Relevant studies were reviewed, assessed for quality, and data were extracted. / Results. Thirty-four studies met the inclusion criteria. There was a suggestion of increasing neurological events with increasing severity and progression of carotid stenosis. There was some evidence for an association with carotid plaque morphology. No consistent association was found with clinical factors, impaired cerebral vasoreactivity, or cerebral embolic signals. / Conclusions. The evidence is insufficient to reliably identify individuals with asymptomatic carotid stenosis who are at a higher risk of cerebral ischemic events.

No description available.

Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Activation of the NO-cGMP pathway is associated with myocardial protection against ischaemia/reperfusion injury. However, high-cholesterol diets alter function of this pathway and these alterations have been implicated in both ischaemic/reperfusion injury and the development of ischaemic heart disease. Little is known about the effects of supplements such as Red Palm Oil (RPO) on the myocardial NO-cGMP-signalling pathway. RPO consists of saturated, mono-unsaturated and poly-unsaturated fatty acids and is rich in antioxidants such as β-carotene and Vitamin E (tocopherols and tocotrienols). The aims of this study were: 1) to determine whether dietary RPO-supplemention protects against ischaemia/reperfusion injury in rats fed a standard rat chow (control) and cholesterol-enriched diets and 2) if so, to investigate possible mechanisms for this protection. Male Long-Evans rats were fed a standard rat chow or a standard rat chow plus cholesterol and/or RPO-supplementation for 6 weeks. Myocardial functional recovery was measured and hearts were freeze-clamped for determination of myocardial phospholipid, cAMP/cGMP concentrations, total myocardial nitric oxide concentrations, lipid hydroperoxide production and superoxide dismutase- and nitric oxide synthase activity in isolated rat hearts subjected to 25 minutes of normothermic total global ischaemia. In addition, the degree of phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal protein kinase (JNK) and protein kinase B (PKB/Akt) was investigated. Furthermore, the effect of RPO-supplementation on caspase-3 activation and poly (ADP-ribose) polymerase (PARP)-cleavage in hearts subjected to ischaemia and reperfusion was also investigated. Our data show that dietary RPO-supplementation protects the hearts of rats on a standard rat chow (control) and hypercholesterolaemic diet against ischaemia/reperfusion injury as reflected by improved aortic output recovery. Increased intracellular cardiomyocyte NO concentrations as observed in control hearts supplemented with RPO after 120 minutes hypoxia may contribute to the elevated cGMP concentration and may confer some of the cardioprotection to the ischaemic/reperfused heart. Although improved functional recovery with RPO-supplementation of a high-cholesterol diet was also associated with an increase in intracellular cardiomyocyte NO production after hypoxia compared to the non-hypoxic conditions, it could not be linked to increased NO-cGMP signalling. These data are in agreement with other studies, which showed that high-cholesterol diet impairs NO-cGMP signalling and confirms our hypothesis that elevated cGMP concentrations may not be the only mechanism of protection. We have also shown that RPOsupplementation caused increased phosphorylation of p38 and PKB, reduced phosphorylation of JNK and attenuation of PARP cleavage, which may contribute to the protection of the cell against apoptosis. Based on our results we propose that the myocardial protection offered by RPO-supplementation of rats on a normal and hypercholesterolaemic diet may be associated with either its antioxidant characteristics and/or changes in the fatty acid composition of the myocardium during ischaemia/reperfusion. Furthermore, we demonstrated for the first time that RPO-supplementation protects the isolated perfused working rat heart during reperfusion from ischaemia/reperfusion-induced injury through a MAPK-dependent pathway. / AFRIKAANSE OPSOMMING: Aktivering van die NO-cGMP sein transduksie pad word geassosieer met miokardiale beskerming teen isgemie/herperfusie skade. Hoë cholesterol diëte verander egter die funksie van die pad en hierdie veranderings speel ‘n rol in beide isgemie/herperfusie besering en die ontwikkeling van isgemiese hartsiekte. Daar is egter min inligting beskikbaar oor die uitwerking van aanvullings soos rooi palm olie (RPO) op die miokardiale NO-cGMP sein transduksie pad. RPO bevat versadigde, mono-onversadigde en poli-onversadigde vetsure en is ryk aan anti-oksidante nl. β-karotene en vitamien E (tokoferole en tokotriënole). Die doelwitte van hierdie studie was: 1) om vas te stel of ‘n RPO-aanvulling beskerming bied teen isgemie/herperfusie besering in rotte wat gevoed is met ‘n standaard rotmengsel (kontrole) en cholesterol-verrykte dieet en 2) indien wel, om moontlike meganismes van beskerming te ondersoek. Long-Evans manlike rotte is vir 6 weke gevoer met ‘n standaard rotmengsel of ‘n standaard rotmengsel plus cholesterol en/of RPO-aanvulling. Miokardiale funksionele herstel is gemeet en harte is gevriesklamp vir die bepaling van miokardiale fosfolipied, cAMP/cGMP, totale stikstofoksied, lipied hidroperoksied, superoksied dismutase en stikstofoksied sintase in geïsoleerde rotharte wat vir 25 minute onderwerp was aan normotermiese totale globale isgemie. Hiermee saam is die graad van fosforilering van ekstrasellulêre sein gereguleerde kinase (ERK), p38 mitogeen-geaktiveerde proteïen kinase (p38 MAPK), c-Jun-N-terminale proteïenkinase (JNK) en proteïen kinase B (PKB/Akt) ondersoek, asook kaspase-3 aktivering en poli (ADP-ribose) polimerase (PARP) kliewing in harte blootgestel aan isgemie en herperfusie. Ons resultate toon dat RPO-aanvulling van rotte op ‘n normale en hipercholesterolemiese dieet die hart beskerm soos getoon deur verbeterde herstel van aortiese uitset. Verhoogde intrasellulêre miokardiale NO vlakke in kontrole harte met ‘n RPO-aanvulling wat blootgestel was aan 120 minute hipoksie, mag bygedra het tot die verhoogde cGMP vlakke en beskerming van die hart tydens isgemie en herperfusie. Alhoewel verbeterde funksionele herstel met RPO-aanvulling van ‘n hoë cholesterol dieet ook geassosieer is met ‘n toename in intrasellulêre miokardiale NO produksie ná hipoksiese toestande, kon dit nie verbind word met verhoogde aktivering van die NOcGMP sein transduksie pad nie. Hierdie resultate stem ooreen met ander studies wat aangetoon het dat hoë-cholesterol diëte die NO-cGMP seinpad onderdruk. Hierdie bevinding bevestig ons hipotese dat verhoogde cGMP vlakke moontlik nie die enigste beskermingsmeganisme is nie. Ons resultate het ook gewys dat RPO-aanvulling fosforilering van p38 en PKB/Akt verhoog, fosforilering van JNK verminder en PARP kliewing onderdruk. Dit dui op beskerming van die sel teen apoptose. Ons resultate dui aan dat die miokardiale beskerming wat RPO-dieet aanvulling bied moontlik geassosieer kan word met sy anti-oksidant eienskap en/of veranderinge in die vetsuur samestelling van die miokardium tydens isgemie/herperfusie. Ons het ook vir die eerste keer bewys dat RPO-aanvulling die geïsoleerde geperfuseerde werkende rothart gedurende herperfusie beskerm teen isgemie/herperfusie besering deur die aktivering en/of deaktivering van die MAPK afhanklike pad.