Ischemic stroke is a devastating cerebrovascular disease resulting in high mortality rate and distressing sequelae such as hemiplegia, ataxia and even visual impairment. Retinal ischemia refers to a common pathological feature shared by many blinding diseases including retinal vascular occlusions, diabetic retinopathy, glaucoma, and retinopathy of prematurity. Ischemia/reperfusion injury is implicated in both of these pathological conditions, which greatly impact on one’s daily life. The eventual consequence of the insult is irreversible neuronal cell death and functional deterioration. Apart from current symptomatic treatment for these diseases, researchers and clinicians are dedicated to look for ideal neuroprotectant to meet the clinical needs. Traditional Chinese medicine has been received accumulating attention in recent years, and Lycium barbarum is one of them. The polysaccharides (LBP) utilized in the present study are the rich extracts of the fruit of Lycium barbarum that has been shown to exert many biological effects. This study aims to evaluate its protective effects in cerebral and retinal ischemia, which has not yet been fully investigated. A well-established rodent model, middle cerebral artery occlusion, was utilized in the present study to mimic cerebral and retinal ischemia/reperfusion injury. In the study of cerebral ischemia, both pre-treatment and post-treatment of LBP were explored. Seven-day LBP pre-treatment revealed significant protection against neurological deficits and cerebral infarction. Besides, it attenuated cerebral edema and glial activation, as well as preserved blood-brain barrier integrity. Further study showed that these beneficial effects of LBP pre-treatment might act via anti-apoptosis, antioxidation and anti-inflammation. However, similar findings were not noted in LBP post-treatment experiments, possibly due to the timing of intervention. In the investigation of retinal ischemia, the observation time was prolonged to 7 days after the insult. Electroretinogram was used to evaluate the functional alternation of retinal neurons. Sustained retinal dysfunction was induced by two-hour ischemia. LBP pre-treatment with continuous daily supplementation effectively alleviated visual dysfunction and protected the retina from morphological impairment including neuronal death, glial activation and blood-retinal barrier disruption. Similarly, these protective effects might be associated with the involvement of attenuation of apoptosis and oxidative stress. In conclusion, LBP pre-treatment with continuous daily supplementation protected the brain and retina, both functionally and morphologically, from ischemia/reperfusion injury. This dosing regimen hold great promise in serving as a prophylactic neuroprotectant in patients at high risk for ischemic stroke, as well as preserving normal visual function and reducing irreversible neuronal death in ischemic retinopathies. Further studies on the active ingredients and underlying mechanisms would be informative for better application of LBP in clinical situation. / published_or_final_version / Ophthalmology / Doctoral / Doctor of Philosophy

Ischemia-reperfusion (IR) injury occurs during myocardial infarction and during some cardiovascular surgeries. Animal studies support the role of endurance exercise training in preventing myocardial IR injury and coronary endothelial dysfunction. In human and animal studies, habitual exercise has been shown to attenuate endothelial dysfunction caused by aging and disease. It is unknown; however, if exercise can protect against vascular IR injury in humans and if so, whether these effects persist with advancing age. Using 20 minutes of forearm ischemia and the response of the brachial artery as a noninvasive surrogate model for the heart, the association between the mode of exercise training (endurance versus resistance) and vascular IR injury was examined in young healthy adults in the first study. Endothelial function, as measured by flow-mediated dilation (FMD) in the brachial artery, decreased significantly after forearm ischemia, suggesting that this noninvasive model of the heart produces significant and measureable vascular injury. These measures returned to baseline levels within 30 minutes following ischemia, illustrating the transient nature of this form of IR injury. The magnitude of injury and recovery from ischemia were not significantly different among young sedentary, endurance-trained, and resistance-trained subjects, suggesting that exercise training is not associated with protection from vascular IR injury in a young, healthy population. In the second study, the association between aging, endurance exercise training, and vascular IR injury was studied. Twenty minutes of forearm ischemia was associated with a transient fall in brachial FMD in young and older sedentary and endurance-trained subjects. Young subjects recovered more quickly from IR injury than older subjects. Within 30 minutes of injury, the endothelial function of the young group was back to baseline while blunted endothelial function persisted in older subjects for greater than 45 minutes after injury. There was no association between endurance exercise training and enhanced recovery from IR injury. These findings suggest that aging is associated with delayed recovery from vascular IR injury and that endurance training does not appear to modulate the vascular IR injury responses. / text

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

The overarching aim of the thesis research was to investigate mechanisms altered by protein-energy malnutrition (PEM), a common stroke co-morbidity factor that could affect the extent of brain damage and recovery following stroke. To model stroke, the rat 2-vessel occlusion model of global brain ischemia was employed. To characterize the effects of PEM, three states of malnutrition were assessed: PEM co-existing with brain ischemia (Study 1), effects of PEM independent of brain ischemia (Study 2), and PEM developing after brain ischemia (Study 3). The first hypothesis tested was co-existing PEM triggers an exacerbated glial response to global brain ischemia. The failure to achieve a consistent model of global ischemia prevented us from drawing conclusions on whether co-existing PEM exacerbates reactive gliosis. Nonetheless, this study demonstrated that mean temperature and temperature fluctuation are increased within the first 24hr of exposure to a low protein diet. The second hypothesis tested was PEM causes sustained changes in core temperature that are associated with an inflammatory response. Exposure to a low protein diet caused an immediate small and transient increase in mean temperature and a larger sustained increase in temperature amplitude. As malnutrition evolved, mean temperature declined. PEM stimulated an acute-phase response, characterized by an increase in the positive acute-phase protein, alpha-2-macroglobulin (A2M), and a decrease in the negative acute-phase protein, albumin. This response appeared to be aberrant, since the positive acute-phase protein, alpha-1-acid glycoprotein (AGP), was decreased with PEM. The final hypothesis tested was PEM developing after global brain ischemia exacerbates systemic and hippocampal inflammation, which is associated with diminished neuroplasticity. The effects of PEM on the acute-phase response are persistent following brain ischemia, as demonstrated by decreased serum albumin and increased serum A2M. A decrease in the positive acute-phase protein, haptoglobin, strengthened the evidence that PEM triggers an atypical reaction. The strong glial response elicited by global ischemia was unaltered by PEM. However, PEM influenced hippocampal neuroplasticity mechanisms, as GAP-43 and synaptophysin were significantly lower at d21. In summary, it has been demonstrated that PEM affects core temperature, the systemic acute-phase reaction and the neuroplasticity response to global brain ischemia.

Η αγγειογένεση είναι η βασική διαδικασία κατά την οποία τα αγγεία αναπτύσσονται από ήδη υπάρχοντα. Αποτελεί βασική προϋπόθεση για ένα μεγάλο αριθμό φυσιολογικών και παθολογικών διαδικασιών. Πρόσφατα καθιερώθηκε ο όρος αρτηριογένεση εστιάζοντας στην διαδικασία με την οποία ένα προϋπάρχον μικρό αρτηριόλιο ωριμάζει και αναδιαμορφώνεται σε ένα μεγαλύτερο αρτηρίδιο που μπορεί να άγει πολύ μεγαλύτερη ποσότητα αίματος στη μονάδα του χρόνου ως απάντηση στην αυξημένη ενδαγγειακή ροή και πίεση αίματος. Έτσι, η ενεργοποίηση της αρτηριογένεσης και η αιμοδυναμική υποβοήθηση των υπαρχόντων παραπλεύρων αγγείων (θεραπευτική αγγειογένεση) αποτελεί σήμερα έναν ελκυστικό ερευνητικό στόχο με σκοπό την επαναιμάτωση και συνεπώς σωτηρία ευαίσθητων στην ισχαιμία ιστών όπως η καρδιά, ο εγκέφαλος και τα άκρα. O τομέας της θεραπευτικής αγγειογένεσης και αρτηριογένεσης αποτελεί σήμερα ένα ταχέως εξελισσόμενο πεδίο στην σύγχρονη έρευνα του καρδιαγγειακού συστήματος. Η στεφανιαία και περιφερική αρτηριακή αθηροσκλήρωση αποτελεί παγκοσμίως την πρωτεύουσα καρδιαγγειακή αιτία θνησιμότητας και νοσηρότητας. Σε πολλούς ασθενείς δεν είναι εφικτή ούτε η διαδερμική ενδοαγγειακή αντιμετώπιση με αγγειοπλαστική και μεταλλικές ενδοπροθέσεις (stents) ούτε η χειρουργική επαναγγείωση του ισχαιμούντος ιστού μέσω αρτηριακής παράκαμψης (bypass). Έτσι η έρευνα για τα καρδιαγγειακά νοσήματα έχει εστιάσει την προσοχή της σε νέους ελάχιστα επεμβατικούς τρόπους επαναιμάτωσης, όπως είναι η θεραπευτική αγγειογένεση με ενδοαρτηριακή, ενδομυϊκή ή περιαγγειακή έγχυση διαφόρων αγγειογενετικών παραγόντων οι οποίοι διεγείρουν τα ενδογενή αγγειογενετικά βιοχημικά μονοπάτια και προάγουν την ανάπτυξη παράπλευρου δικτύου (αρτηριογένεση) ικανού να αναστρέψει την ιστική ισχαιμία και υποξία. Διάφοροι αγγειογενετικοί παράγοντες έχουν δοκιμαστεί στην κλινική πράξη για την ενίσχυση του παράπλευρου αρτηριδιακού δικτύου στην στεφανιαία νόσο και στην περιφερική αγγειοπάθεια με αμφιλεγόμενα μέχρι τώρα αποτελέσματα. Η θρομβίνη είναι ένα ένζυμο με δραστικότητα σερινοπρωτεάσης που αποτελεί μόριο κλειδί στους βιοχημικούς καταρράκτες της πήξης και αιμόστασης. Πρόσφατες έρευνες έχουν αποδείξει επίσης ότι η θρομβίνη ενεργοποιεί την διαδικασία της αγγειογένεσης μέσω μηχανισμών εξαρτώμενων και ανεξάρτητων από την πήξη του αίματος. Πρόσφατα βρέθηκε ότι η θρομβίνη συμμετέχει στις διεργασίες επιβίωσης των ενδοθηλιακών κυττάρων. Αυτό μπορεί να ανοίξει νέους ορίζοντες όσον αφορά το γενικότερο ρόλο της θρομβίνης στην αγγειοπροστασία και τη συμβολή της στη διατήρηση της ακεραιότητας του τοιχώματος των αγγείων. Η αλληλεπίδραση των αγγειογενετικών αυξητικών παράγοντων και των διαφόρων μηχανισμών αγγειοπροστασίας είναι ουσιώδης τόσο για την ανάπτυξη όσο και για την ωρίμανση των νέων αιμοφόρων αγγείων κατά την εξέλιξη των φαινομένων της αγγειογένεσης και της αρτηριογένεσης. Σκοπός της συγκεκριμένης ερευνητικής δουλειάς ήταν η δοκιμασία, επιβεβαίωση, και κατά κύριο λόγο η σύγκριση της αγγειογενετικής δραστηριότητας της θρομβίνης με άλλους καταξιωμένους αυξητικούς παράγοντες σε ένα in vivo μοντέλο ισχαιμίας κάτω άκρων, με απώτερο στόχο την εφαρμογή της για την πρόκληση θεραπευτικής αγγειογένεσης και επαναιμάτωσης στα πλαίσια κλινικών συνδρόμων ισχαιμίας. Παράλληλα, αναπτύχθηκαν και εφαρμόστηκαν καινοτόμες πειραματικές μεθοδολογίες για την πρόκληση, μορφολογική απεικόνιση και λειτουργική μελέτη της αγγειογένεσης και αρτηριογένεσης στα κάτω άκρα κονίκλων. Μελετήσαμε τις αρτηριογενετικές ιδιότητες της θρομβίνης σε ένα μοντέλο αμφοτερόπλευρης ισχαιμίας κάτω άκρων κονίκλου και δείξαμε για πρώτη φορά ότι η εξωγενής χορήγηση θρομβίνης ενισχύει το σχηματισμό νέων λειτουργικών και σταθερών παράπλευρων αγγειακών δικτύων και αποκαθιστά την αιματική άρδευση των ίσχαιμων κάτω άκρων. Η παρούσα μελέτη παρέχει τα πρώτα επιστημονικά δεδομένα για την αρτηριογενετική δράση της θρομβίνης, η οποία πιθανόν υπερέχει άλλων κλασσικών αγγειογενετικών παραγόντων (bFGF, VEGF). Επιπλέον εξετάσαμε διάφορα δοσολογικά σχήματα χορήγησης του μορίου της θρομβίνης και επιβεβαιώσαμε την αγγειογενετική της δράση τόσο σε ένα χειρουργικό, όσο και σε ένα ελάχιστα επεμβατικό ενδοαγγειακό μοντέλο αμφοτερόπλευρης ισχαιμίας των οπίσθιων άκρων του κονίκλου. Η επιτυχία της επαναιμάτωσης με θρομβίνη επαληθεύτηκε τόσο μορφολογικά με ψηφιακή αγγειογραφία όσο και λειτουργικά με σύγχρονα πρωτόκολλα υπολογιστικής τομογραφίας αιματώσεως. / Compared with angiogenesis, arteriogenesis is a distinct process based on the remodeling and maturation of pre-existing arterioles into large conductance arteries. Therapeutic angiogenesis has been proposed as a potential treatment for ischemic atherosclerotic diseases. Since a variety of angiogenic factors have been tested with inconsistent so far clinical results, the challenge remains in identifying the factor(s) that will stimulate functional neovascularization. Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by regulating and organizing a network of angiogenic mediators. Also, it was recently demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, providing evidence on a potential role of thrombin in vascular protection and maintenance of vessel integrity. Based on these observations, we hypothesized that thrombin may promote the development of mature functional blood vessels. We have shown that thrombin promoted the formation of large collateral vessels and improved the perfusion of distal ischemic tissue in a rabbit hindlimb ischemia model. These results provide new insights in understanding the involvement of thrombin in vascular formation and point to a novel role of thrombin in arteriogenesis. Interplay between angiogenic growth factors and vascular maturation mechanisms are essential for the cascade of reactions involved in arteriogenesis, i.e. development of large conductance collateral vessels that may adequately compensate for atherosclerotic arterial occlusions. Thrombin stimulated robust collateral networks in the ischemic limbs, which was associated with a significant recovery of ischemic tissue perfusion as assessed by in-vivo perfusion studies. This may provide the basis for applications of thrombin and its nonthrombogenic analogs in therapeutic angiogenesis.

Ischaemic heart disease (IHD), which is a Chronic Disease of Lifestyle, has been rated as one of the key illnesses that have progressively materialised as a threat across the wide demographic spectrum of South Africa’s population. Internationally, literature is relatively scarce on the psychosocial consequences of the disease for the ill person, but limited information does exist on these consequences as they affect South African individuals and their families. This study sought firstly to understand the experience of persons with IHD, their partners and medical practitioners and secondly how they coped with the illness. Participants were selected with the assistance of cardiologists at a private hospital in KwaZulu-Natal. Theoretical sampling determined the number of persons who participated in the study. Data was collected using in-depth interviews in accordance with the qualitative descriptive design and narrative inquiry that underpinned the research. Participants (13) were determined once data-saturation was reached. The data revealed that spousal support and religion/spirituality played important roles in helping people manage the illness. Genetics and lifestyle choices contributed to participants’ IHD. Not recognising their symptoms, owing to misinformation by the medical fraternity, contributed to participants not realising they were having a heart attack. Religious/spiritual coping mechanisms were among those mentioned by participants, and depression (not clinically assessed), anxiety and sadness were noted among the consequences for them. Two of the cardiologists did not acknowledge depression; nevertheless research studies have linked depression with major cardiac attacks and to surgery following cardiac problems. The data points to the lack of communication between participants and their health practitioners and the belief held by participants that the medical personnel were not particularly interested in their psychosocial wellbeing. This seems to be borne out by the limited data obtained from health practitioners. Social workers can play important roles in the health team by ensuring that the psychosocial needs of persons experiencing IHD and their families are attended to. Social workers can publish in health journals to make the medical fraternity aware of the contribution that social workers skills can make to persons who are ill. / Thesis (M.A.)-University of KwaZulu-Natal, Durban, 2012.

Many populations have low intakes of n-3 fatty acids, yet there is substantial evidence that the long chain n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3), found at high concentrations in the brain, is required for the proper development of the nervous system. However, less is known about requirements of long chain n-3 fatty acids for maintenance and function of the nervous system in later life. Several recent studies have reported that high amounts of long chain n-3 fatty acids reduce the extent of brain damage caused by cerebral ischemia in animals. However, whether or not a dietary deficiency of n-3 fatty acids increases the extent of injury when cerebral ischemia occurs has not been previously reported. The present studies, therefore, sought to determine if a diet deficient in n-3 fatty acids influences the extent of brain injury in the rat following cerebral ischemia. Male rats were fed an n-3 fatty acid adequate (control), an n-3 fatty acid deficient, or a high DHA diet for 5 weeks from weaning. Middle cerebral artery occlusion (MCAO) was induced and infarct volume was measured by 2,3,5,-triphenyltetrazolium chloride staining 24 hours after the procedure. Brain and platelet fatty acids were analyzed by gas liquid chromatography. DHA (22:6n-3) was 21-28% lower in brain phospholipids, and 17% lower in brain total fatty acids in the n-3 fatty acid deficient compared to control group, while 22:6n-3 was 12% higher in total brain fatty acids in the high DHA group than the control group. There was no significant difference in infarct volume (203, 220 and 218 mm³) among the control, n-3 fatty acid deficient, and high DHA groups, respectively. Platelet fatty acids and platelet aggregation were assessed to determine if these were influenced by the high DHA diet, and could possibly explain the observation of an apparent, but not statistically significant, higher number of rats with hemorrhages in the high DHA diet group. Platelet lipid arachidonic acid was not lower and platelet aggregation, assessed ex vivo using whole blood with a platelet function analyzer, was not longer in rats fed the high DHA compared to control or n-3 fatty acid deficient diets. In summary, dietary n-3 fatty acid deficiency did not increase the extent of brain injury following cerebral ischemia. The possibility that high dietary 22:6n-3 might increase susceptibility to cerebral hemorrhage will require further study.

EGFR TKIs are standard therapy for advanced NSCLC. In order to define their role in early disease, we implemented a phase II trial of neoadjuvant gefitinib in clinical stage I NSCLC. Tumour shrinkage was seen in 43% of patients, with 11% achieving RECIST partial response (PR). Analysis of molecular markers showed EGFR TKD mutations in 17% of cases, being the only associated with PR. For the first time we defined the histopathological response of NSCLC to these agents, characterized by reduction in tumour cellularity and proliferative index as well as presence of non-mucinous BAC histology. Clinical PR tumours also presented large areas of stromal fibrosis with presence of focal residual tumour. In a characterization of intracellular signalling response, EGFR dephosphorylation in the residues Y1068 and Y1173 was not concordant and only the former was significantly reduced. pAkt Ser473/Akt and Thr308/Akt ratios were significantly reduced but observed among both, clinical responders and resistant patients. Interestingly, reduction in pEGFR Y1068 was significantly associated with increase in tumour cellularity (p=0.047), Ki-67 index (p=0.018) and tumour growth (p=0.019) with a residual perinuclear localization been detected, suggesting a novel mechanism of resistance involving receptor internalization. Finally, we determined that the EGFR protein remains stable up to one hour of post resection ischemia but two to three tumour samples are necessary for an adequate tumour representation. Furthermore, EGFR cytoplasmic compartment presented the best association with clinical response in our cohort. Taking all together, we were able to generate the first clinical trial exploring the use of an EGFR TKI in early NSCLC, characterizing for the first time the histopathological and signalling responses to these agents with an evidence of a potential novel mechanism of resistance. Finally, we observed that multiple samples collection for an adequate tumour representation, and assessment of the cytoplasmic compartment, are warrant.

Computer-aided drug design is an exceptionally useful tool for screening a large number of potential drug molecules to evaluate their therapeutic potential. This technique is both effective and economical. Approximately 120 imidazole-containing molecules were computationally designed and evaluated using gas-phase and solution-phase simulations to assess their propensity for acting as a chelating agent with twenty-six biologically relevant cations. Of particular interest was their ability to chelate Zn2+ and Ca2+, which play a key role in the degradation of neurons following an ischemic stroke. The ultimate goal was to design a small molecule that could be administered before a medical procedure that featured stroke as a possible side effect. In the event that a stroke occurred, the destruction of neurons caused by release of excess Ca2+ and Zn2+ would be diminished and the patient would maintain motor and cognitive function. Promising in silico results were obtained.