Cerebral ischaemia by mechanism of thrombosis is one of the leading causes of disability and/or death worldwide, the outcome thereof increasing in severity with advancing age. Cerebral ischaemia triggers a cascade of events including inflammation, blood-brain barrier disruption and apoptosis. It is well known that oestrogen is neuroprotective through various mechanisms including the interruption of inflammation, regulation of thrombosis and delay of apoptosis. This creates a strong factorial interconnection in predicting the consequences of cerebral ischaemia. Since platelets have a central role in thrombosis and inflammation, their ultrastructure being altered in conditions of inflammatory and thrombotic derivation, the question arises whether chemical analysis of coagulation factors and ultrastructural analyses of platelet morphology may provide further insight into the role of oestrogen during ischaemic insult associated with stroke. Accordingly, an exclusively hyperglycaemic modification of the two-vessel occlusion model for inducing experimental cerebral ischaemia was established, since pre-ischaemic hyperglycaemia is known to intensify the outcome of cerebral ischaemic injury. Consequent neural tissue injury levels were correlated for three experimental groups (males, cyclic and acyclic females) of Sprague Dawley rats at vital times, to the presence of oestrogen as well as changes in coagulation factors and ultrastructure. This design allowed for an association to be formed between cerebral ischaemia, inflammation and thrombotic potential. Collectively the results strongly suggest that oestrogen is indeed neuroprotective through various actions including roles in the regulation of thrombosis and inflammation, targeting neural cells through the inhibition of apoptosis and exerting anti-inflammatory and antioxidant effects. It is evident that under the influence of oestrogen in cyclic females, there is reduced neural tissue injury as well as a lesser degree of inflammation evident in coagulation factor analysis and platelet activation morphology when compared to males and acyclic females. Oestrogen therefore exerts positive effects on the outcome of cerebral ischaemia through mechanisms which regulate inflammation, thrombosis and apoptosis. Furthermore it is unmistakeable that neural injury is closely shadowed, if not preceded, by inflammatory changes in the coagulation system, particularly manifested in platelet ultrastructure. It is therefore suggested that platelets may be used successfully to follow the progression of events of cerebral ischaemia and possibly assist in the assessment of treatment strategies and their effects on haemostasis. This research advances the understanding that inflammation is evident soon after ischaemic insult and if such inflammation is not curbed, necrosis of platelets and more severe injury to neural tissue may follow. Therefore, the development of agents which not only target thrombosis, but also which control inflammation must be explored to advance treatment strategies. It is proposed that even before it is determined whether a stroke has been caused by thromboembolism or haemorrhage; it will be beneficial to immediately target inflammation in order to prevent most severe consequences in human patients. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Anatomy / unrestricted

Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors that mediate various actions of Acetylcholine (ACh) in the central nervous system and peripheral nervous system. In mammals, five distinct mAChR subtypes (M1-M5) have been recognised with the M2 subtype being predominantly present in the heart. The mAChR antagonists are routinely used for the treatment of various pathophysiological conditions including respiratory conditions. However, it has been postulated that mAChR antagonists may increase morbidity and mortality in chronic obstructive pulmonary disorder (COPD) and asthma patients with underlying cardiovascular disease, raising concerns regarding the cardiovascular safety of these agents. The current study was therefore undertaken to investigate the effects of individual mAChR antagonists in the setting of myocardial ischaemia reperfusion injury and oxidative stress models. We also investigated whether the inhibition of the mitochondrial permeability transition pore (MPTP) with cyclosporine-A (CsA) in the presence and absence of individual mAChR antagonists provided protection against ischaemia reperfusion injury. Furthermore, we also aimed to investigate the intracellular signalling pathway associated with mAChRs antagonists mediated myocardial injury under the stress conditions. Langendorff results showed that the non-selective M1-M3 mAChR antagonist, ipratropium bromide, the M2 mAChR antagonist, AF-DX 116 and the M3 mAChR antagonist, DAU 5884 significantly increased the infarct size to risk ratio of the heart in conditions of ischaemia and reperfusion. Detrimental effects of AF-DX 116 and DAU 5884 were abrogated by co-treatment of these drugs with mAChR agonist, acetylcholine (ACh) and/or CsA. Cell viability data of isolated cardiac myocytes revealed that AF-DX 116 and DAU 5884 caused a concentration dependent decrease in the viability of cardiac myocytes as well as causing a reduction in the time taken to depolarisation and hypercontracture under oxidative stress. AF-DX 116 and DAU 5884 significantly increased the levels of p-SAPK/JNK and decreased the levels of p-Akt and p-ERK. In addition, ACh and CsA showed to activate p-Akt and p-ERK. To conclude, the data suggest that AF-DX 116 and DAU 5884 caused cardiotoxicity at cellular, tissue and protein level in conditions of ischaemia reperfusion injury and oxidative stress. Furthermore, inhibition of the mitochondrial transition pore with CsA protected against the AF-DX 116 and DAU 5884 induced injury via activation of the pro-survival proteins, p-Akt and p-ERK.

Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Toll-like receptor (TLR)-mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre-treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)-induced protection was lost in TLR3 knockout mice. In poly (I:C)-treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF-κB activation in the brain. Poly (I:C) also decreased I/R-induced apoptosis by attenuation of Fas/FasL-mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase-3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)-induced interaction between Fas and FADD as well as caspase-3 and -8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co-association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase-3 and -8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.

Aims Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling. Methods and results Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN-or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3β (glycogen synthase kinase-3β), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice. Conclusion These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism.

Aims: Bifunctional apoptosis regulator (BAR) is an endoplasmic reticulum protein that interacts with both the extrinsic and intrinsic apoptosis pathways. We hypothesize that over-expression of BARΔRING prevents apoptosis and injury following ischaemia/reperfusion (I/R) and attenuates doxorubicin (DOX)-induced cardiotoxicity. Methods and results: We generated a line of transgenic mice that carried a human BARΔRING transgene under the control of the mouse α-myosin heavy chain promoter. The RING domain, which binds ubiquitin conjugating enzymes, was deleted to prevent auto-ubiquitination of BAR and allow accumulation of the BAR protein, which binds apoptosis-regulating proteins. High levels of human BARΔRING transcripts and 42 KDa BARΔRING protein were expressed in the hearts of transgenic mice. When excised hearts were reperfused ex vivo for 45 min as Langendorff preparations after 45 min of global ischaemia, the functional recovery of the hearts, expressed as left ventricular developed pressure x heart rate, was 23 ± 1.7% in the non-transgenic hearts compared with 51.5 ± 4.3% in the transgenic hearts (P < 0.05). For in vivo studies, mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by 4 h of reperfusion. The infarct sizes following I/R injury, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (29 ± 4 vs. 55 ± 4%, P < 0.05). In hearts of mice subjected to cardiac I/R injury, BAR transgenic hearts had significantly fewer in situ oligo-ligation-positive cardiac cells (5.0 ± 0.4 vs. 13.4 ± 0.5%, P < 0.05). Over-expression of BARΔRING also significantly attenuated DOX-induced cardiac dysfunction and apoptosis. Conclusion: Our results demonstrate that over-expression of BARΔRING renders the heart more resistant to I/R injury and DOX-induced cardiotoxicity, and this protection correlates with reduced cardiomyocyte apoptosis.

Objective:Iatrogenic acute limb ischaemia in paediatric patients is a well-recognised complication of vascular access. This retrospective review of a paediatric intensive care unit identified patients who developed iatrogenic acute limb ischaemia between January 2008 and July 2013. Methods: The medical records of inpatients diagnosed with acute limb ischaemia during the study period were reviewed. Patients with other causes of acute limb ischaemia were excluded. A descriptive analysis of demographics, primary diagnosis, type of vascular access used, affected anatomical region, clinical presentation, type of therapy, type of block, response to intervention used and outcomes was conducted. Results:A total of 28 patients presented with signs of acute limb ischaemia, of whom 28.6% were aged <30 days, 46.4 % were between one and 12 months and 25% were between one and five years old; 78.6% of the affected limbs were lower limbs. Four patients had resolution of ischaemia upon removal of the vascular access devices. 23 patients received various forms of pharmacological sympathectomy, in addition to conservative therapy. One patient had missing data on the type of sympathectomy that was done. The response to the sympathectomies was: 60.9% good, 8.7% moderate, 8.7% poor and in 21.7% no responses. Documented tissue loss related to the ischaemia occurred in six (21.4%) of the 28 patients. Conclusions: Iatrogenic acute limb ischaemia in children are usually managed without surgical intervention. Pharmacological sympathectomies lead to increased blood flow to the affected limb via vasodilatation of collateral vessels, with an added advantage of reducing ischemic pain. The improved blood flow is postulated to avoid and/or minimise the amount of tissue loss. Pharmacological sympathectomies may, thus, have a role to play in th e management of iatrogenic acute limb ischaemia in the paediatric population.

Introduction The neuropsychological deterioration after cardiac surgery involving cardiopulmonary bypass (CPB), is ascribed to brain ischaemia caused by, amongst others, hypotension, cerebral hyperthermia, cerebral embolism, interaction between pharmacological methods and hypothermia during ischaemia, and the directly neurotoxic of anaesthetic drugs. AimTo investigate the effect of the anaesthetic techniques midazolam plus ketamine (MK) or sufentanil (S) on the quantitative EEG (QEEG), reaction time (RT), serum neuron specific enolase (NSE), and serum S-100<font face="symbol">b</font> protein after cardiac surgery involving CPB in humans. Patients and methodsThe sample consisted of a total of 42 patients scheduled for elective coronary artery bypass (CABG) or valve replacement (VR). All patients were not available for all the postoperative tests. Patients were allocated randomly to group MK or S. Anaesthetic technique: S or MK. Isoflurane was administered when necessary. CPB technique: 30°C, membrane oxygenation, a 40 <font face="symbol">m</font>m filter in the arterial cannula and <font face="symbol">a</font>-stat-blood gas management, blood pressure of 50 to 70 mm Hg and a haematocrit <font face="symbol">></font> 22%. Patients were weaned from CPB when nasopharyngeal temperature reached a maximum of 37,5°C. QEEG and RT was performed 1 to 2 days preoperatively and 5 to 6 days postoperatively.Serum-NSE and -S-100<font face="symbol">b</font> protein were measured preoperatively, 2 minutes after going on CPB, after rewarming to 37°C, just before the end of CPB and 2, 4, 10, 20, 30, and 48 hours after CPB. ResultsQEEG: The most noticable finding was an increase in slow wave activity (relative <font face="symbol">q</font> and <font face="symbol">s</font>). The QEEG outcome was better after CABG than after VR (p < 0,001), but not different between MK and S (p = 0,5000). <font face="symbol">Dq</font>% was better with MK than S (p = 0,0120). <font face="symbol">Dq</font>% (p = 0,0010), <font face="symbol">Da/q</font>% (p = 0,0090) and <font face="symbol">D</font>PS% (p = 0,0025) was better after CABG than VR. Reaction time: There was a significant deterioration in 5/18 (27,78%) of MK and 12/18 (66,67%) of S (p = 0,0220). The change in accuracy in sequential reation time 1 (p = 0,0100), and sequential reation time 2 (p = 0,0970) and the cumulative accuracy was better with MK than S(p = 0,0020). Chemical markers: Over groups 14,8% of patients had a poor NSE and 61,9% a poor S-100<font face="symbol">b</font> outcome. Within groups a poor NSE outcome was found in 14,8% of MK and 14,8% of S (p = 1,0000), and 4,8% of CABG but 23,8% of VR (p = 0,1840). Within groups as adverse S-100<font face="symbol">b</font> outcome was found in 42,9% of MK but 81,0% van S (p = 0,0250; Fisher's exact test), and 66,7% of CABG and 57,1% of KV (p = 0,7510. According to area under the curve of corrected NSE, CABG had a better outcome than VR (p = 0,0040). According to both maximum S-100<font face="symbol">b</font> level and the area under the curve of S-100<font face="symbol">b</font>, an interaction occurred between the anaesthetic technique and the procedure, with VR doing better with MK while CABG did significantly better with S (p = 0,0180 en 0,0040 respectively). Conclusion, shortcomings, significance and contribution This study has shown that, in as far as brain damage is concerned, the outcome was probably better with MK than with S, and CABG better than VR. An interaction was found between the anaesthetic technique and the type of operation. / Thesis (MD)--University of Pretoria, 2003. / Anaesthesiology / unrestricted

No / Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in ¿-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12¿15-fold increase in ¿7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the ¿4 nAChR subunit was not altered. These data point to the ¿7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating ¿7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).

Cerebral ischaemia is a leading cause of disability and death world-wide. The only effective treatments are thrombolytic therapy (plasminogen activator; tPA) and hypothermia (33?C). However, tPA has limited clinical application due to its short therapeutic time window and its specific application in thrombo-embolic stroke. Moderate hypothermia (33?C) is only being used following cardiac arrest in comatose survivors. Hence more treatments are urgently required. The first step in developing new treatments is the identification and characterisation of a potential therapeutic target. Since brain damage following cerebral ischaemia is associated with disturbances in intracellular calcium homeostasis, the sodium-calcium exchanger (NCX) is a potential therapeutic target due to its ability to regulate intracellular calcium. Currently, however there is uncertainty as to whether the plasma membrane NCX has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue I compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3-/-) and wild-type mice (Ncx3+/+) following global cerebral ischaemia. In order to perform this study I first established a bilateral common carotid occlusion (BCCAO) model of global ischaemia in wild-type C57/BlHsnD mice using controlled ventilation. After trials of several ischaemic time points, 17 minutes was established as the optimum duration of ischaemia to produce selective hippocampal CA1 neuronal loss in the wild-type mice. I then subjected NCX3 knockout and wild-type mice to 17 minutes of ischaemia. Following the 17 minute period of ischaemia, wild-type mice exhibited 80% CA1 neuronal loss and 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed > 95% CA1 neuronal loss and 95% CA2 neuronal loss. Following experiments using a 17 minute duration of global ischaemia, a 15 minute duration of ischaemia was also evaluated. Wild-type mice exposed to a 15 minute period of ischaemia, did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed 45% CA1 neuronal loss and 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient for the NCX3 protein are more susceptible to global cerebral ischaemia than wild-type mice. My findings showing a neuroprotective role for NCX3 following ischaemia, suggest that the exchanger has a positive role in maintaining neuronal intracellular calcium homeostasis. When this function is disrupted, neurons are more susceptible to calcium deregulation, with resultant cell death via calcium mediated pathways. Therefore, improving NCX activity following cerebral ischaemia may provide a therapeutic strategy to reduce neuronal death.

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.