The research was designed to investigate the effects of AIT-082, a derivative of the purine hypoxanthine containing a para-amino benzoic acid moiety, on neural cells. AIT-082 has been shown to possess a number of neurotrophic and neuroprotective properties and to enhance memory. Furthermore, AIT-082 is undergoing clinical trials as a potential treatment for Alzheimers disease.<p>The first part of the study investigated the ability of AIT-082 to influence cellular ATP levels in cortical neurons. Decreased energy metabolism is a key point in Yings (Ying, 1996a) theory of the development of Alzheimers disease. Previous work with AIT-082 had shown that it could protect hippocampal neurons from cellular damage caused by sublethal doses of glutamate. Specifically, AIT-082 prevented neurite degeneration. Also, AIT-082 was shown to increase mitochondrial membrane potential, especially at the distal tips of the neurites, in hippocampal neurons. I hypothesized that AIT-082 was protecting the neurons by increasing the ability of the mitochondria to generate ATP and thereby increasing the amount of ATP available to the cell. ATP was collected and measured from cortical neuron cultures that were exposed to glutamate, AIT-082, glutamate and AIT-082. The ATP levels were compared to the ATP levels from cortical neuron cultures that were exposed to vehicle for glutamate and AIT-082. The results did not significantly increase ATP levels in cortical neurons following glutamate exposure. <p>The next set of experiments involved investigations into the ability of AIT-082 to influence phosphorylation events in neural cells. AIT-082 shares some neurotrophic and neuroprotective properties with a group of drugs called the immunophilin ligands. The neuroprotective properties of the immunophilin ligands are mainly due to their ability to influence protein phosphorylation by inhibiting the activity of calcineurin a protein phosphatase. The first set of experiments used western blot techniques to measure serine peptide and threonine peptide phosphorylation levels in proteins from whole brain homogenates that were incubated with vehicle, AIT-082, and GMP. Both AIT-082 and GMP caused an increase in the level of serine peptide phosphorylation compared to vehicle but only the increase caused by GMP treatment proved to be significant. Further, threonine phosphorylation levels were significantly increased by GMP but not AIT-082. Phosphorylation levels of short peptide sequences containing either a phosphorylated serine or threonine residue were also measured in neuronal and astrocytic cultures. The neuronal cultures were exposed to 4 h of hypoxia to mimic the conditions of reduced energy availability observed in Alzheimers disease brains. Astrocyte cultures were exposed to 4 h of hypoxia/ischemia for the same reason. Both cell types were allowed to recover for 0, 1, 4, 12 and 24 hours with or without AIT-082 following the insult. AIT-082 treatment did not significantly affect phosphorylation levels of proteins harvested from either neuron or astrocyte cultures at any time period. I conclude therefore, that AIT-082 is not able to influence phosphorylation of the short amino acid sequences containing phosphorylated serine or threonine residues that could be detected by the primary antibodies used in my experiments.

Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

The endothelium is an integral mediator of vascular homeostasis and a dysfunctional endothelium is now recognized as an early marker of atherosclerosis. Importantly, the non-invasive measurement of endothelial function by flow-mediated dilation (FMD) predicts future cardiovascular events. However, the appropriate method of its assessment and the mechanisms that govern FMD are still poorly understood. We investigated alternative parameters and methods of FMD measurement in healthy volunteers and cardiovascular disease patients. We found time to peak FMD to be highly variable both within and between individuals. Accordingly, continuous arterial diameter measurement post-cuff release was more sensitive in discriminating between health and disease compared to the measurement of diameter at 60’’ post-cuff release. Reperfusion to an ischemic tissue can paradoxically contribute to endothelial dysfunction development and further tissue damage, in a phenomenon known as ischemia and reperfusion (IR) injury. Previous exposure to sublethal ischemia (ischemic preconditioning (IPC)) can reduce sensitivity to IR injury and pharmacologic agents have since been shown to mimic this response. Using the FMD technique, we investigated various preconditioning strategies to prevent IR-induced endothelial dysfunction in the forearm vasculature of healthy volunteers. The sodium-hydrogen exchanger inhibitor amiloride and the angiotensin-converting enzyme inhibitor captopril were found not to provide endothelial protection from IR. In contrast, potent protection from IR-induced endothelial dysfunction was observed during the high-estrogen, late follicular phase of the menstrual cycle in pre-menopausal women. Finally, daily episodes of IPC were found to provide endothelial protection equipotent to an acute episode of IPC. The findings from the FMD methodological study highlight the importance of continuous arterial diameter measurement post-cuff deflation, and provide mechanistic insight that may contribute to measurement standardization and normalization. The results of the preconditioning studies improve our understanding of potential approaches to mitigate the detrimental effects of IR on the endothelium in humans.

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.

Tesi realitzada al Centre d' Investigació Cardiovascular (CSIC-ICCC), de l' Hospital de la Santa Creu i Sant Pau de Barcelona / Las alteraciones del metabolismo lipídico causadas por la isquemia miocárdica tienen profundos efectos en el estado fisiológico del corazón. En el corazón isquémico, la hipoxia incrementa la formación de vacuolas lipídicas en zonas periféricas al área de riesgo al aumentar la síntesis endógena de triglicéridos y reducir la beta-oxidación de ácidos grasos. A su vez, durante la hipoxia, las células musculares lisas de la pared vascular (VSMC) tienen una captación incrementada de lipoproteínas de baja densidad (LDL). Se ha descrito un efecto sinérgico entre la hipoxia y la acumulación de lipoproteínas en la inducción de genes inflamatorios y genes involucrados en el metabolismo lipídico. Los estudios realizados in vitro e in vivo demuestran que el receptor de LDL (LDLR) no es el responsable de la captación incrementada de colesterol en células vasculares sometidas a hipoxia pues está regulado positivamente por los SREBP de tal forma que la acumulación de colesterol inhibe su expresión. Sin embargo el receptor LRP1 está regulado negativamente por los SREBP por lo que, independientemente de los niveles de colesterol intracelular, es capaz de captar el colesterol de las lipoproteínas y sobreacumularlo en la célula. Además el LRP1 está sobreexpresado por la hipoxia en las VSMC de las arterias coronarias y puede explicar mecanísticamente los efectos sinérgicos entre hipoxia e hipercolesterolemia en la acumulación lipídica intracelular. Actualmente se desconoce el papel del LRP1 en el cardiomiocito y las consecuencias de las alteraciones en su expresión sobre el metabolismo lipídico. Se ha descrito que la lipoprotein lipasa presente en la superficie de los cardiomiocitos media la captación de lipoproteinas utilizando un receptor no identificado hasta el momento para la captación selectiva de colesterol. Por otro lado, se sabe que el LRP1 media la captación selectiva de colesterol por células vasculares. Todos estos resultados sugieren que el LRP1 podría participar en la captación de lípido por el cardiomiocito, y dejan entrever la importancia de los mecanismos moleculares involucrados en su modulación. En primera instancia los objetivos fueron analizar el efecto de la hipoxia en la expresión del receptor LRP1 en cardiomiocitos neonatales de rata y HL-1, así como los mecanismos involucrados en este efecto. También determinar el papel del LRP1 en la captación de VLDL en condiciones de hipoxia por los cardiomiocitos. Nuestros resultados, publicados en el artículo “Low-density lipoprotein receptor-related protein 1 mediates hypoxia-induced very low density lipoprotein-cholesteryl ester uptake and accumulation in cardiomyocytes”, demostraron que la hipoxia incrementa la expresión del LRP1 a través del factor inducible por hipoxia HIF-1α, y ésta sobreexpresión induce la captación y acumulación de colesterol esterificado procedente de VLDL en cardiomiocitos. A continuación estudiamos el efecto de dosis hipercolesterolémicas de LDL e hipertrigliceridémicas de VLDL en la expresión del LRP1 en cardiomiocitos, así como la correlación entre la expresión del LRP1 y la acumulación lipídica en el ventrículo izquierdo de pacientes con cardiomiopatía isquémica. Publicados en el artículo “Low-density lipoprotein receptor-related protein 1 expression correlates with cholesteryl ester accumulation in the myocardium of ischemic cardiomyopathy patients” nuestros resultados sugieren que la regulación al alza del LRP1 juega un papel primordial en la acumulación de ésteres de colesterol en el miocardio de corazones isquémicos y esto puede suponer un “target” con el fin de prevenir efectos deletéreos de esta acumulación en la cardiomiopatía isquémica. Por último, en el tercer artículo, “Low-density lipoproteins promote unstable calcium handling linked to reduced SERCA2 and connexin-40 expression in cardiomyocytes”, observamos que el colesterol procedente de LDL desestabiliza el manejo del calcio por parte de cardiomiocitos HL-1 al reducir la expresión de SERCA2 y la velocidad de conducción del calcio por la Conexina40. / "Role of the low density lipoprotein receptor-related protein-1 (LRP1) in ischemia and cardiovascular pathophysiology" Currently, it is unknown the role of LRP1 in the cardiomyocyte and the consequences of its changes in expression on lipid metabolism. It has been reported that lipoprotein lipase, present on the surface of cardiomyocytes, mediates the uptake of lipoproteins using an unidentified receptor so far for the selective uptake of cholesterol. Furthermore, it is known that the LRP1 mediates the selective uptake of cholesterol by vascular cells. All these results suggest that LRP1 may be involved in lipid uptake and accumulation by the cardiomyocyte and show the importance of understanding the molecular mechanisms involved in the modulation of LRP1 by hypoxia. In the first instance the objectives were to analyze the effect of hypoxia on LRP1 receptor expression in neonatal rat cardiomyocytes and HL-1, and the mechanisms involved in this effect. Also determine the role of LRP1 in VLDL uptake under hypoxia by cardiomyocytes. Our findings, published in the article "Low-density lipoprotein receptor-related protein 1 mediates hypoxia-induced very low density lipoprotein-cholesteryl ester uptake and accumulation in cardiomyocytes", showed that hypoxia increases LRP1 expression through transcription factor HIF-1α, and this overexpression induces the uptake and accumulation of cholesteryl esters from VLDL in cardiomyocytes. Then, we estudied the effect of LDL and VLDL in the LRP1 expression, and the correlation between LRP1 expression and lipid accumulation in the left ventricle of patients with ischemic cardiomyopathy. Published in the article "Low-density lipoprotein receptor-related protein 1 Expression Correlates with cholesteryl ester accumulation in the myocardium of ischemic cardiomyopathy patients" our results suggested that the LRP1 upregulation plays a role in cholesterol accumulation in the ischemic myocardium and this could be a "target" in order to prevent deleterious effects of this accumulation. Finally we analyzed the effects of LDL on calcium handling by cardiomyocytes HL-1. In the third article, "Low-Density Lipoproteins Promote unstable linked to calcium handling Reduced SERCA2 and connexin-40 expression in cardiomyocytes", we observed that colesterol from LDL destabilizes calcium handling by cardiomyocytes HL-1 reducing SERCA2 expression and calcium conduction velocity by Connexin40.

Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.

Our laboratory previously found that protein-energy malnutrition (PEM) existing prior to brain ischemia impaired functional outcome measured in an open field test, and one-third of animals showed a marked increase in reactive gliosis. It was hypothesized that PEM worsened stroke outcome by increasing inflammation via increased activation of the transcription factor, nuclear factor kappa B (NFκB). Mongolian gerbils (11-12 wk old) were randomly assigned to a control diet (12.5% protein) or a protein-deficient diet (2%) for 28 days. The control group on average gained 4.9g and the PEM group lost 7.4g. PEM gerbils had significantly decreased food intake (P<0.001; unpaired t-test). Animals were then subjected to global ischemia or sham surgery, resulting in four experimental groups. Global ischemia was achieved by a 5 min bilateral common carotid artery occlusion with tympanic temperature regulated at 36.5 ± 0.2C. PEM independently increased hippocampal NFκB activation by three times higher than control diet animals at 6hr after surgery (p=0.014; 2-factor ANOVA) detected by electrophoretic mobility shift assay (EMSA). There was no significant effect of ischemia on NFκB activation and there was no interaction of diet and ischemia. Serum glucose and serum cortisol were also measured since both variables can be affected by PEM and can influence stroke outcome, but there was no significant effect of diet or ischemia. Because of the increased NFκB activation observed in PEM-Sham animals, a second experiment investigated if PEM also increased NFκB activation in the absence of surgery. Gerbils of the same age were randomly assigned to either control diet or PEM for 28 days but did not receive any surgery. PEM consistently increased NFκB activation. Since PEM exists in 16% of elderly stroke patients at admission, the data suggest that PEM may worsen stroke outcome through increased activation of NFκB. Because increased NFκB activation was also observed in PEM independent of ischemia, the data also have implications for the inflammatory response of protein-energy malnourished elderly in general.

Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

<p>In a mouse model of ischemic stroke, infarct volume is highly variable and strain dependent, but the natural genetic determinants responsible for this difference remain unknown. To identify genetic determinants regulating ischemic neuronal damage and to dissect apart the role of individual genes and physiological mechanisms in infarction in mice, we performed forward genetic mapping analyses of surgically induced cerebral infarct volume. We have identified multiple quantitative trait loci (QTL) that modulate infarct volume, with a major locus (<italic>Civq1 </italic>) on chromosome 7 accounting for over 50% of the variation, with a combined LOD score of 21.7. Measurement of infarct volume in chromosome substitution strains (CSS) and two additional intercrosses validate that <italic>Civq1</italic> on chromosome 7 is present in multiple inbred strains. Interval-specific ancestral SNP haplotype analysis for <italic>Civq1</italic> results in 5 candidate genes. A causative gene underlying <italic>Civq1</italic> may regulate collateral artery formation and genetic variations in the gene may result in the differential outcome of cerebral infarction. Additionally, we have identified a locus of large effect, <italic>Civq4</italic>, modulating infarct volume through a mechanism different from collateral circulation. In conclusion, the extent of ischemic tissue damage after distal middle cerebral artery occlusion (MCAO) in inbred strains of mice is regulated by genetic variation mapping to at least 4 different loci. A single locus on chromosome 7 determines the majority of the observed variation in the trait in multiple mouse strains. <italic>Civq1</italic> appears to be identical to <italic>Lsq1</italic>, a locus conferring limb salvage and reperfusion in hindlimb ischemia. The identification of the genes underlying these loci may uncover novel genetic and physiological pathways that modulate cerebral infarction and provide new targets for therapeutic intervention in ischemic stroke, and possibly other human vascular occlusive diseases.</p> / Dissertation

Objective: Ischemic spinal cord injury is a serious complication of aortic surgery. The mechanism underlying ischemic preconditioning (IPC) protection against spinal cord ischemia/reperfusion (I/R) injury is unclear. We investigated the role of spinal cord autoregulation in tolerance to spinal cord I/R injury induced by IPC. Although the extracellular signal-regulated kinases 1 and 2 (ERK1/2) are generally regarded as related to cell survival and proliferation, increasing evidence suggests that the role of the ERK1/2 pathway in I/R injury is contributory to inflammation. We investigated the effect of blocking ERK1/2 pathway to inhibit inflammation reaction in tolerance to spinal cord I/R injury. Methods: In the part 1 study, Sprague-Dawley rats were randomly assigned to 4 groups. IPC (P) group animals received IPC by temporary thoracic aortic occlusion (AO) with a 2-F Fogarty arterial embolectomy catheter for 3 min. I/R injury (I/R) group animals were treated with blood withdrawal and temporary AO for 12 min, and shed blood reinfusion at the end of the procedures. (P+I/R) group animals received IPC, followed by 5 min reperfusion, and then I/R procedures for 12 min. Sham (S) group animals received anesthesia and underwent surgical preparation only. Neurological functions were evaluated, and lumbar segments were harvested for histopathological examination. To evaluate the role of autoregulation in IPC, spinal cord blood flow and tissue oxygenation were continuously monitored throughout the procedure duration. In the part 2 study, spinal cord ischemia rats was induced by occluding the thoracic descending aorta with a balloon catheter introduced through a femoral artery, accompanied by concomitant exsanguinations. Rats in the control group were given dimethyl sulfoxide (vehicle) before undergoing spinal cord ischemia/reperfusion injury. In the U0126-treated group, rats were pretreated with an inhibitor of ERK1/2, U0126, to inhibit ERK1/2 phosphorylation. The sham rats underwent aortic catheterization without occlusion. Parameters, including neurologic status, neuronal survival, inflammatory cell infiltration, and interleukin-1£] production in the spinal cords, were compared between groups. Results: The Tarlov scores in the (I/R) group were significantly lower than those in the (S), (P), and (P+I/R) groups on days 1, 3, 5, and 7. The numbers of surviving motor neurons in the (S), (P), and (P+I/R) groups were significantly higher than those in the (I/R) group. The (P) group exhibited higher spinal cord blood flow and tissue oxygenation after reperfusion than the (S) group. The (P+I/R) group exhibited higher spinal cord blood flow and tissue oxygenation within the first 60 min after reperfusion than the (I/R) groups. In the part 2 study, early ERK1/2 phosphorylation was observed after injury in the control group, followed by abundant microglial accumulation in the infarct area and increased interleukin-1£] expression. In the U0126 group, U0126 treatment completely blocked ERK1/2 phosphorylation. Microglial activation and spinal cord interleukin-1£] levels were significantly reduced. Neuronal survival and functional performance were improved. Conclusions: IPC ameliorates spinal cord I/R injury in rats, probably mediated by triggering spinal cord autoregulation and improving local spinal cord blood flow and tissue oxygenation. The ERK1/2 pathway may play a noxious role in spinal cord ischemia/reperfusion injury by participating in inflammatory reactions and cytokine production. According to our findings, these concepts may be the new therapeutic targets in patients requiring aortic surgery.