Sepsis, a great challenge to the physician, is characterized with massive oxidative stress of tissue, cytokine inflammation and increases in nitric oxide (NO) production. Meanwhile, free radical induced by oxidative stress also injures cell membrane or DNA. The way to terminate free radical chain reaction is to administer antioxidant. The commonly used anesthetic, propofol, was thought to be with antioxidant capacity. In the first part of this thesis, we investigated the different role of oxidative injury and NO via systemic injection of LPS in rats. We demonstrated oxidative injury is associated with both early and late stage whereas NO is engaged primarily in late stage cardiovascular depression. Propofol, a rapid onset and fast recovery anesthetic, is attributed to protect anainst cardiovascular depression via attenuating the late stage NO surge in aorta by inhibition of iNOS upregulation. We also examine the influence of propofol on temporal changes in power density of frequency components of systemic arterial pressure (SAP) variability in rat with sepsis and the role of inducible NO synthase (iNOS). We have the conclusions that iNOS-induced NO might be involved in the manifestation of high-frequency and low-frequency components of the SAP spectrum during endotoxemia when low-dose propofol is used and the effect of NO is blunted when high-dose propofol is administered. Due to further investigation was needed to the cellular protective mechanisms of propofol, we delineate the effect of propofol to free radical related enzymel involved in sepsis via both in vivo and vitro studies with rats subjected to LPS (15 mg/kg) and H9C2, L2, NR8383 (derived from rat cardiac myocyte, lung, macrophage, respectively), respectively. Our results demonstrated that propofol may play the major protective role on iNOS, superoxide dismutase and p47 phox oxidative enzymes on lung epithelial cells. Propofol also provided protective effects on cardiac myocyte and macrophage with suppression of iNOS only although free radical production were all significantly suppressed. Ischemia-reperfusion (IR) injury may also produce a lot of free radical and cytokines to cause tissue damage and is common in clinical. We investigated the effect of propofol on free radical and cytokine production via this different model and compared with another rapid recovery anesthesitc, sevoflurane. Aortic decalmping surgery in porcine and their monocyte, aortic and coronary smooth muscle cells were applied for in vivo and in vitro model, respectively. We also demonstrated that propofol but not sevoflurane suppressed the production of free radical and cytokine in monocyte and smooth muscle cells but not in vivo model. In sepsis and IR model that produced a lot free radical and cytokines, propofol eliminated the free redical and cytokines via suppressed different kinds of oxidative enzymes in different cells of different organs to express its protective role. However, as an anesthetic, propofol must be used carefully to perform its maximal benefit.

According to the theory of Traditional Chinese Medicine, the main etiology of stroke results from blood stasis. Ligustic Rhizoma (LR), a Chinese herb, is considered to stimulate stasis-dispelling. As Tetramethyl Pyrazine (TMP) is a major component of LR, the aim of the present study is to investigate the effects of TMP on cerebral infarct. We establish an animal model of cerebral infarct by occluding the both common carotid arteries and right middle cerebral artery for 90 minutes, then reperfusing for 24 hrs. Effects of TMP on cerebral infarct are evaluated by the ratio of infarction areas and modified neurologica severity scale (mNSS). In addition, we observe the changes of ED1, tumor necrosis factor-£\ (TNF-£\) and interleukin-1£] (IL-1£]) immuno-reacting cells in the infarction region. The founding indicate that pre-treatment with TMP 100 mg/kg, 120 mg/kg and 140 mg/kg, and post-treatment with TMP 100 mg/kg will decrease the ratio of cerebral infarction area and the neurological deficit. Moreover, pre-treatment TMP 100 mg/kg also decreases ED1, TNF-£\ and IL-1£] immuno-reacting cell. In conclusion, TMP can decrease cerebral infarction area and neurological deficit. The effects of TMP, at least in part, are closely related to microglia, TNF-£\ and IL-1£], suggesting that TMP can be used to treat stroke in human.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成5年9月14日 加藤亨嗣氏の博士論文として提出された

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Centre for Neuroscience. Title from pdf file main screen (viewed on January 27, 2010). Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

The peripheral immune system plays a role in delayed neural injury after stroke. This response originates from the spleen as splenectomy prior to middle cerebral artery occlusion (MCAO) in rats significantly reduces infarct volume in the brain. This research is based on the hypothesis that inhibiting the splenic response will reduce neurodegeneration after stroke. Studies in animals have implicated lymphocytes as the immune cell type that is detrimental following MCAO. Interferon gamma (IFNγ) has been identified as a pro-inflammatory cytokine that is also detrimental following stroke. IFNγ is important because it activates microglia and macrophages in a pro-inflammatory nature that increases neural injury following stroke. Therefore IFNγ was examined in the brain and the spleen following MCAO. IFNγ protein was elevated at 24 h in the spleen and at 72 h in the brain post MCAO. Microglia/macrophages become maximally activated at 72 h in the brain after MCAO. Splenectomy decreases the levels of IFNγ in the brain following MCAO. Systemic administration of IFNγ reversed the protective effects of splenectomy. The cellular response to MCAO was examined next because of the difference in time between the spike in IFNγ in the spleen and the delayed increase in the brain. The cellular response from the spleen was studied by labeling splenocytes five days prior to MCAO with a fluorescein dye. Tissues were examined 48 and 96 h post MCAO or sham MCAO for fluorescence. These cells were released from the spleen into circulation at 48 h post MCAO and migrated to the brain where the cells produced IFNγ at 96 h post MCAO. IFNγ appears to play a role in the splenic response to stroke. One protein that is up regulated by cells that have been activated by IFNγ, interferon-inducible protein 10 (IP-10) is part of the inflammatory cycle driven by IFNγ. IP-10 recruits more IFNγ producing T helper (Th) cells to the site of injury. IP-10 has the unique ability to attract Th1 cells, the pro-inflammatory Th cells, and inhibit Th2 cells, the anti-inflammatory Th cells. This leads to more IFNγ production as IFNγ is the signature cytokine of a Th1 response. IP-10 is significantly increased in the brain at 72 h post MCAO, similar to IFNγ expression. In the spleen IP-10 increased at 24 h and remained elevated out to 96 h following MCAO. IFNγ signaling was inhibited by utilizing an IFNγ neutralizing antibody administered beginning 24 h post MCAO. The IFNγ antibody treated group had decreased infarct volumes, IP-10 levels in the brain, and appeared to have decreased T cells in the ipsilateral hemisphere at 96 h post MCAO. Following ischemic stroke splenocytes are released into circulation and migrate to the brain. They release IFNγ to activate microglia/macrophages in a proinflammatory phenotype causing an increase in IP-10 levels. IP-10 then potentiates the Th1 driven inflammation which inhibits the Th2 response. The elevated levels of IFNγ increase neural injury following MCAO. Blocking IFNγ selectively blocks the inflammatory facet of the immune response to reduce stroke induced neurodegeneration. This leaves the other immune responses intact and able to contribute to tissue repair, regeneration, and able to respond to infections. Selectively inhibiting IFNγ signaling is a promising stroke therapeutic.

Stroke is the fifth leading cause of death in the United States and a major cause of long-term disability in industrialized countries. The core region of an ischemic stroke dies within minutes due to activation of necrotic pathways. Outside of this core region is the penumbral zone, where some perfusion is maintained via collateral arteries. Delayed cell death occurs in this area due to the triggering of apoptotic mechanisms, which expands the ischemic injury over time. The cellular and molecular events that produce the expansion of the ischemic core continue to be poorly understood. The increases in the amyloid precursor protein and pathogenic secretases lead to the increase in amyloid-β (Aβ) production. The relatively small amount of research in this area has hampered development of stroke therapy designed to prevent neuronal and glial cell degeneration in the penumbra. Currently, there is a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (≥ 4.5 hr post-stroke). Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury in vitro following ischemia, both of which have been shown to play important roles following an ischemic stroke. Treatment with afobazole decreased microglial activation in response to ATP and Aβ, as indicated by reduced membrane ruffling and cell migration. Prolonged exposure of microglia to ischemia or Aβ conditions resulted in glial cell death that was associated with increased expression of the pro-apoptotic protein, Bax, the death protease, caspase-3 and a reduced expression in Bcl-2. Co-application of afobazole decreased the number of cells expressing both Bax and caspase-3, while increasing the cells expressing Bcl-2 resulting in a concomitant enhancement in cell survival. While afobazole inhibited activation of microglia cells by Aβ25-35, it preserved normal functional responses in these cells following exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24 hr exposure to Aβ25-35. However, co-incubation with afobazole returned these responses to near control levels. Therefore, stimulation of sigma-1 and sigma-2 receptors by afobazole prevents Aβ25-35 activation of microglia and inhibits Aβ25-35-associated cytotoxicity. Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that ischemia or Aβ results in an increased expression of the pro-apoptotic protein Bax and the death protease caspase-3, while at the same time decreasing expression of the anti-apoptotic protein, Bcl-2. However, unlike observations made with microglia, afobazole was unable to modulate this ischemia-induced expression, but was able to modulate Aβ-induced expression of apoptotic proteins while still rescuing neurons from death. Additional experiments were carried out to understand this disparity between the failures of afobazole to prevent the up-regulation of pro-apoptotic genes while retaining the ability to mitigate neuronal death. Although the neurons were still alive they were in a senescent state and were unresponsive to depolarization by high K+. However, these findings are still positive due to the ability of afobazole to delay neuron death, thus minimalizing the toxic environment of the penumbra. These comorbidities of ischemia and Aβ toxicity may lead to potentiated responses and increase the risk for various vascular dementias. It was of particular interest to study how the convergence of ischemia, acidosis and Aβ influence cellular activity and survival within core and penumbral regions. Application of Aβ increased the [Ca2+]i overload produced by concurrent ischemia + acidosis application in isolated cortical neurons. We found that the acid-sensing ion channels 1a (ASIC1a) are involved in the potentiation of [Ca2+]i overload induced by Aβ. Furthermore, afobazole (100 uM) abolished Aβ potentiation of ischemia + acidosis evoked [Ca2+]i overload, which may represent a therapeutic strategy for mitigating injury produced by Aβ and stroke.

published_or_final_version / Anaesthesiology / Master / Master of Philosophy

Stroke is a devastating, neurological dysfunction due to brain blood supply disturbance. It is responsible for increasingly high rate of mortality and disability worldwide. This thesis comprises two original studies involving 868 patients at risk of ischemic stroke and/or hemorrhagic stroke. The first study investigated aspirin’s effect among patients with intracranial hemorrhage. Unlike Caucasians which hemorrhagic strokes account for 10-15% of all strokes; in Chinese, intracranial hemorrhages strike up to 35%. After such, anti-platelet agent like aspirin is often avoided for fear of recurrent intracranial hemorrhages, despite compelling indications. However, clinical data is limited. In this single-centered observational study, we included 440 consecutive Chinese patients with a first spontaneous intracranial hemorrhage surviving the first month performed during 1996-2010. 56 patients (12.7%) of these 440 patients were prescribed aspirin after intracranial hemorrhage (312 patient-aspirin years). After a mean follow-up of 62.2 ± 1.8 months, 47 patients had recurrent intracranial hemorrhage(10.7%, 20.6 per 1,000 patient years). Patients prescribed aspirin did not have higher risk of recurrent intracranial hemorrhage compared with those without (22.7 per 1,000 patient-aspirin years vs. 22.4 per 1,000 patient years, p=0.70). Multivariate analysis identified age > 60 years and hypertension as independent predictors for recurrent intracranial hemorrhage. In a subgroup analysis: the incidence of combined vascular events including recurrent intracranial hemorrhage, ischemic stroke, and acute coronary syndrome was statistically lower in patients prescribed aspirin than without (52.4 per 1,000 patient-aspirin years, vs. 112.8 per 1,000 patient-years, p=0.04). Implications of the results: despite having a substantial risk for recurrent intracranial hemorrhage, post-intracranial hemorrhage ones are at risk for thrombotic vascular events and management goal should thus focus on ameliorating overall cardiovascular risk instead of preventing recurrent intracranial hemorrhage. Hence, thrombo-prophylaxis should still be considered. The second study investigated the relation between premature atrial complexes and new-onset atrial fibrillation together with other cardiovascular events. Premature atrial complexes though taken as benign phenomenon, are common in patients with underlying conditions such as coronary heart disease, chronic rheumatic heart disease. While prompt management of atrial fibrillation may prevent ischemic stroke, atrial fibrillation is often unfound until ischemic stroke occurs. In this study, 428 patients without atrial fibrillation but complained of palpitations, dizziness or syncope were recruited. 107 patients with >100 premature atrial complexes/day were defined to have frequent premature atrial complexes. After a mean follow-up of 6.1 ±1.3 years, 31 patients (29%) with frequent premature atrial complexes developed atrial fibrillation compared with 29 patients (9%) with premature atrial complexes?100/day (p<0.01). Cox regression analysis revealed: frequent premature atrial complexes, age>75 years and coronary artery disease were independent predictors. In secondary endpoint (ischemic stroke, congestive heart failure, and death), patients with frequent premature atrial complexes were more at risk than those without (34.5% vs. 19.3%) (Hazard ratio: 1.95, 95% confidence interval: 1.37-3.50, p=0.001). Cox regression analysis showed: age> 75 years, coronary artery disease and frequent premature atrial complexes were independent predictors. These permit early identification of high risks patients of new atrial fibrillation and other events, thus promoting appropriate preventive treatment. / published_or_final_version / Medicine / Master / Master of Philosophy

 Ischemic stroke is the leading cause of death and disability in human diseases all around the world. As effective treatment for ischemic stroke is still absent, seeking for new therapy is of great interest. Currently, several key pathological cascades following cerebral ischemia have been explored to develop further therapies. Among them, reactive nitrogen species (RNS) has been indicated to play a critical role in cerebral ischemia reperfusion injury. As one of the RNS, peroxynitrite contributes to the neural cell death and subsequent brain dysfunction in the process. Thus, development of antioxidants targeting on peroxynitrite could be an important strategy for the treatment of cerebral ischemia-reperfusion injury. Baicalin is a polyphenolic compound isolated from roots of Scutellaria baicalensis. Baicalin exerted protective effects against cerebral ischemia-reperfusion injury but the mechanisms are not clear yet. In this study, we investigated the free radical scavenging ability and neuroprotective effects of baicalin. According to our results, baicalin neutralized DPPH radicals effectively. By using electron paramagnetic resonance (EPR) spin trapping technology and fluorescent probe DAF-2DA, we found that baicalin dose-dependently scavenged superoxide, but had very low effect on elimination of nitric oxide. The immunofluoresent results revealed that baicalin at the concentration of 50 M completely suppressed the nitrotyrosine formation induced by 3-morpholinylsydnoneimine chloride (SIN-1, a peroxynitrite donor) in neuroblastoma SH-SY5Y cells. Mass spetrum provided direct evidence of the peroxynitrite scavenging ability of baicalin. Using MTT assays, we found that baicalin totally reversed peroxynitrite-induced cytotoxicity in SH-SY5Y cells and protected SH-SY5Y cells in oxygen glucose deprivation (OGD) and following reoxygenation injury. Furthermore, in vivo experiments revealed that intravenous injection of baicalin exerted better neuroprotective effect than intraperitoneal administration in rats underwent middle cerebral artery occlusion (MCAO). After cerebral ischemia reperfusion, rats treated with 3 mg/kg of peroxynitrite decomposition catalyst (FeTMPyP) or 25 mg/kg of baicalin revealed a smaller size of infarction volume, suppressed neural cell death and reduced nitrotyrosine formation than MCAO rats. However, baicalin did not alter the expression of tight junction proteins, claudin-5 and ZO-1, in brain endothelial bEnd3 cell line treated with OGD following reoxygenation. In cerebral ischemia reperfusion rats, administration of FeTMPyP at the dosage of 3 mg/kg diminished the Evans blue leakage caused by blood brain barrier disruption, whereas treatment of baicalin did not show significant effect. In conclusion, this study suggests that baicalin can scavenge peroxynitrite and protect neural cells from peroxynitrite-induced injury. Furthermore, baicalin could prevent brains from cerebral ischemia-reperfusion injury and the neuroprotective mechanisms are associated with the scavenging effects on peroxynitrite. These findings provide new insights into the antioxidant and neuroprotective properties of baicalin and indicate the potential application of baicalin for the treatment of ischemic stroke. / published_or_final_version / Chinese Medicine / Master / Master of Philosophy