Spontaneous and evoked potentials (EPs) were recorded with cross-cortical microelectrode arrays following partial occlusion of the MCA and ACA in urethane-anaesthetised rats. The control group received no occlusion, while the treatment group was injected with anti-stroke peptide Tat-NR2B9c 5min before ischemia. Spontaneous EEG power significantly decreased in the stroke-only group when compared to controls (p<0.001). A greater loss of EEG power was observed on anterior electrodes closer to the occluded area versus posterior contacts in stroke-only rats (p<0.05). The Tat-NR2B9c+stroke group lost significantly less power when compared to stroke-only animals (p<0.05). EP amplitude in the stroke-only group was significantly reduced following ischemia when compared to control and Tat-NR2B9c+stroke animals (p<0.001). Epileptiform discharges were observed in 8/10 untreated stroke rats and 3/5 stroke rats treated with Tat-NR2B9c. The characteristic features of spontaneous and evoked potentials validate this rat focal stroke model for in vivo testing of pharmacological agents.

Current knowledge indicates that nonthermal muscle metaboreflex activity plays a critical role in the modulation of skin vasodilation and sweating. However, the mechanisms of control have primarily been studied during isometric handgrip exercise in which muscle metaboreceptor activation is induced by a brief post-exercise ischemia of the upper limb. While the reflex increase in mean arterial pressure associated with this period of ischemia is consistent with the activation of muscle metaboreceptors, the change in baroreflex activity may in itself modulate the response. Thus, we sought to understand how these nonthermal stimuli interact in modulating the control of skin perfusion and sweating under conditions of elevated hyperthermia. Furthermore, we examined the mechanisms responsible for the maintenance of arterial blood pressure under varying levels of heat stress during isometric handgrip exercise. Our study findings indicate that the parallel activation of muscle metaboreceptors and baroreceptors during post-exercise ischemia causes divergent influences on the control of skin blood flow and sweating; and these nonthermal stimuli are dependent on the level of hyperthermia. Moreover, we report that heat stress reduces the increase in arterial blood pressure during isometric handgrip exercise and this attenuation is attributed to a blunted increase in peripheral resistance, since cardiac output increased to similar levels for all heat stress conditions. These results provide important insight and understanding into the role of muscle metabo- and baroreflex activity on the control of skin blood flow and sweating; along with further knowledge into the cardiovascular mechanisms responsible for the regulation of arterial blood pressure during hyperthermia.

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Mode of access: World Wide Web. / Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 116-154). / Electronic reproduction. / Also available by subscription via World Wide Web / xvii, 157 leaves, bound ill. 29 cm

La isquèmia/reperfusió (IR) intestinal és una patologia clínica amb una elevada mortalitat del 70% que no ha disminuït durant dècades. La IR intestinal succeeix quan hi ha una disminució o una reducció total del flux sanguini cap a l’intestí, seguida pel restabliment d’aquest flux o reperfusió. Aquesta situació pot succeir per diferents causes, entre elles per trombosi, aneurismes o diverses intervencions quirúrgiques, essent la més característica el transplantament intestinal. El procés d’isquèmia/reperfusió provoca alteracions en el metabolisme cel•lular, entre elles el pas de metabolisme aeròbic a anaeròbic i la conseqüent formació de radicals lliures de l’oxigen (ROS). A més, en els neutròfils, s’ha descrit que una IR provoca un retard en la seva mort cel•lular programada o apoptosi, tot i que en el cas concret de la IR intestinal aquesta situació no ha estat descrita. Una correcta apoptosi dels neutròfisl permet que el seu contingut tòxic intracel•lular no provoqui danys en els teixits circumdants, doncs aquestes restes són fagocitades pels macròfags, comportant una millora de la inflamació. Un retard en l’apoptosi condueix a un augment dels ROS y a la incorrecta resolució de la inflamació degut a l’alteració dels mecanismes fagocítics. L’objectiu del nostre estudi és aconseguir una millora o reducció de la inflamació associada a la IR intestinal mitjançant la modulació del retard de l’apoptosi del neutròfil. Per a aquest fi s’han realitzat experiments en models animals de rata (soques Sprague Dowley i Brown Norway) a les que se’ls hi ha realitzat una IR intestinal mitjançant el clampatge de l’artèria mesentèrica. Per a la modulació de l’apoptosi dels neutròfils es va administrar intravenosament Escherichia coli morta i opsonitzada. El mecanisme apoptòtic dels neutròfils s’ha estudiat amb un model anòxic in vitro amb la línia cel•lular MPRO clon 2.1. Aquesta línia cel•lular és capaç de diferenciar-se a neutròfils madurs en presència de factor de creixement (GM-CSF) i àcid retinòic. Els resultats obtinguts en aquest treball demostren que l’apoptosi dels neutròfils es troba retardada després d’una IR intestinal, i que la modulació d’aquesta apoptosi és capaç de millorar el dany associat. La disminució de l’oxigen provoca l’estabilització del Factor Induïble per la Hipòxia (HIF) en els neutròfils, el que porta a una disminució de la via de senyalització WNT i del seu gen diana 24p3, provocant el retard en l’apoptosi d’aquestes cèl•lules. / Intestinal ischemia/reperfusion (IR) injury is a clinical pathology with a high mortality rate that has not diminished through decades. Intestinal IR occurs when there is a blood flow diminution or total reduction through the intestine, followed by the restoration of this flow or reperfusion. This situation can happen because of many causes, between them, thrombosis, aneurisms or different surgical interventions, being the most characteristic of them intestinal transplantation. Ischemia/reperfusion process provokes alterations in the cellular metabolism, between them, the transition from aerobic metabolism to anaerobic and the consequent formation of Reactive Oxygen Species (ROS). Furthermore, in neutrophils, it has been described that IR provokes a delay in their apoptosis, although it has not been described in the intestine yet. A delay in neutrophil apoptosis drives to an increase of ROS and the incorrect resolution of the inflammation because the phagocytic mechanisms are altered. On the contrary, correct neutrophil apoptosis avoids the possibility of damaging the surrounding tissue because their citotoxic content is phagocytosed by macrophages, driving to an amelioration of the inflammation The aim of our work is to obtain an improvement or a reduction of the intestinal IR associated inflammation through the modulation of the neutrophil apoptosis delay. For this end, experiments were conducted in rats (Sprague Dowley and Brown Norway), and the intestinal IR was provoked through the obstruction of the mesenteric artery with ac clamp. For the neutrophil apoptosis modulation, death and opsonized Escherichia coli were intravenously administered. Neutrophil apoptotic mechanism was studied in an in vitro anoxic model (MPRO clon 2.1 cellular line). This cellular line is able to differentiate into mature neutrophils in presence of growth factor (GM-CSF) and retinoic acid. The results obtained in this work demonstrate that neutrophil apoptosis is delayed after an intestinal IR, and that the modulation of this apoptosis is able to ameliorate the associated injury. The decrease of oxygen provokes the stabilization of the Hypoxia Inducible Factor (HIF) in neutrophils, what drives to a decrease in the WNT signaling pathway and its target gene 24p3, provoking a delay in the apoptosis of these cells.

Subsequent to an hypoxic-ischaemic (HI)-insult a multi-faceted complex cascade of events occurs that ultimately results in cellular and neurological impairments within cortical and sub-cortical central nervous system (CNS) regions. In the present studies a modified �Levine� rat-pup model of HI (left carotid artery ligation + 1 hour global hypoxia on post-natal day (PND) 26) was employed to assess the neuroprotective properties of clomethiazole (CMZ; a γ-aminobutyric acid (GABA)A receptor agonist). In this study, histological and electrophysiological paradigms were used to assess the long-term neuroprotective properties of CMZ (414mg/kg/day via mini-pumps). Key enzymes involved in inflammation, namely nitric oxide synthase (NOS) and arginase, were also examined to assess other potential CMZ mechanisms. Assessments were carried out 3- and 90-days post-HI, with extensive ipsilateral CNS lesions evident at a gross histological level, at both the early and long-term stages, with CMZ significantly decreasing the lesion size at 3- and 90-days (P<0.01; P<0.05). Evoked field potential analyses were used to assess hippocampal CA1 neuronal activity ex vivo. Electrophysiological measurements contralateral to the occlusion revealed impaired neuronal function following HI relative to short- and long-term controls (P<0.001, 3- and 14-days; P<0.01, 90-days), with CMZ providing near complete protection (P<0.001 at 3- and 14-days; P<0.01 at 90-days). Both inducible NOS (iNOS) and arginase activities were significantly increased at 3-days (P<0.01), with arginase activity remaining elevated at 90-days post-HI (P<0.05) ipsilaterally. CMZ suppressed the HI-induced increase in iNOS and arginase activities (P<0.001; P<0.05). These data provide evidence of long-term functional neuroprotection afforded by CMZ in a model of HI-induced neurodegeneration. In addition, under conditions of HI, functional deficits were not restricted to the ipsilateral hemisphere and were due, at least in part, to changes in the activity of NOS and arginase. Underlying mitochondrial dysfunction is eminently present in many neuropathological conditions. The full extent of mitochondrial dysfunction in cortical, hippocampal and cerebellar tissues was assessed following HI. Assessment of mitochondrial FAD-linked respiration at both 1- and 3-days post-HI revealed a significant decrease in activity from ipsilateral cortical and hippocampal regions (P<0.001). In addition, significant changes in respiratory function were also evident in contralateral regions and cerebellum, 3-days post-HI (P<0.05). Assessment of the mitochondrial electron transport chain (complexes I-V) and mitochondrial markers of integrity (citrate synthase) and oxidative stress (aconitase) confirmed ipsilateral mitochondrial impairment following HI. Complexes I, II-III, V and citrate synthase were also impaired, in contralateral regions and cerebellum, 3-days post-HI. CMZ treatment provided significant protection to all mitochondrial aspects of neuronal tissue assessed. This study provides evidence of the full extent of mitochondrial damage following an HI-insult and may contribute, in part, to the impairment seen contralaterally. In addition, protection afforded by CMZ extends to preservation of mitochondrial function and integrity. Cerebral ischaemia-induced angiogenesis has been shown within and around infarcted regions and may contribute to a more favourable neurological outcome. The level of angiogenesis was examined using platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31). CD31 immunolabelling 7-days post-HI revealed a significant increase in angiogenesis compared with non-intervention controls (P<0.001). Treatment with CMZ decreased the level of angiogenesis compared to HI + saline (P<0.001) back to non-intervention control levels. Conversely, N[omega]-nitro-L-arginine methyl ester (L-NAME) treatment (5mg/kg/day) exacerbated the ischaemic lesion (P<0.001) and resulted in a marked decrease in angiogenesis compared to non-intervention controls (P<0.001). The extent of cerebral infarction in these studies is dependent on the level of NOS activity with CMZ increasing total NOS levels compared to HI + saline, while L-NAME halted the HI-induce increase in total NOS activity (P<0.001). These results show for the first time, that angiogenesis may be used as an assessment of neurodegeneration / neuroprotection in pathologies of cerebral ischaemia and are directly correlated with changes in NOS activity. These studies have therefore shown that following HI, damage also occurs contralateral to the occlusion, and is not restricted to the ipsilateral hemisphere. In addition, the neuroprotective effects of CMZ have been shown to extend out to 90-days post-HI, whereby significant protection to CA1 neuronal activity was seen. These studies also provide in vivo evidence that CMZ may also afford neuroprotection via anti-inflammatory pathways, as evidenced by a decrease in iNOS and arginase activities. Furthermore, these studies have also show evidence that angiogenesis (CD31) can be used as a diagnostic tool to assess neuroprotection / neurodegeneration.

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p&lt0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p&lt0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p&lt0.001), leading to a 60% survival rate at 48 hours IRI (p&lt0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p&lt0.001), with a survival benefit of 75% (p&lt0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p&gt0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.