京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22040号 / 医博第4525号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 伊佐 正, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Neurons and the underlying vascular structure that maintains the nutrients necessary for their normal function are intrinsically linked. The relationship between neural activity and its accompanying blood flow is called neurovascular coupling. Our understanding of the intricacies of this relationship has evolved over the years from one of pure supply and demand to one that is highly complex and involves various cell types. While the exact mechanisms underlying neurovascular coupling still remains unresolved, altered coupling has been implicated in a variety of pathological conditions. The overall motivation of this thesis was to uncover how specific perturbations to either the neural or vascular system affect the resulting interplay between them. Our hope is that the results could act as a framework for guiding more specific mechanistic dissections in the future.Until recently, technological constraints have precluded the ability to comprehensively characterize neurovascular coupling on a large scale. Much of our understanding of the coupling relationship on a circuit level has been inferred from individual measurements of either neuronal firing or blood flow dynamics. Our lab has the ability to study coupling more directly through simultaneous imaging of both neural and hemodynamic activity. In this thesis, I set out to characterize how coupling could be differentially altered at a mesoscopic level by specifically perturbing either blood flow or cortical circuit organization. Thus, this work is split into two projects. The first investigates the downstream effects of an acute ischemic injury and the second focuses on how a developmental change in neuronal circuit structure alters function. My work in the acute ischemia model allowed us to capture a curious phenomenon called cortical spreading depolarization (CSD). CSDs have been implicated in a range of acute brain injuries, including ischemia. Despite being a neural event, CSDs have a profound impact on the cerebrovascular. Unfortunately, existing work in this field has been discordant and the results have been difficult to interpret. We used wide-field optical mapping to characterize the dynamics and impact of ischemia-triggered CSDs. Our imaging technique revealed that CSDs had a spatially heterogeneous impact on tissue depending on factors such as baseline metabolic condition and spatiotemporal properties of the CSDs themselves. Furthermore, we observed that CSDs were not isolated events and that multiple could occur in succession in a short period of time. By tracking each and every CSD, we were able to characterize the cumulative effects of CSDs on tissue oxygenation. Our results provide a contextual framework that reconciles some of the observed experimental variabilities. We conclude that an ischemic insult triggers a CSD and consequently, a combination of CSD dynamics and the tissue’s metabolic condition begets more CSDs. This pushes the brain deeper into a feedback loop of exacerbating damage. The second study was done in collaboration with Dr. Ewoud Schmidt and Dr. Franck Polleux, and looks at the functional changes mediated by expression of a human-specific gene duplication, SRGAP2C. The human brain exhibits unique features that enable its enhanced cognitive abilities. The Polleux lab found that humanized SRGAP2C mice showed similar features that characterize the human brain, such as increased synaptic density and delayed synaptic maturation. This ultimately led to increased local and long-range cortico-cortical connectivity and even improved the behavioral performance in a texture discrimination task. Thus, we were motivated to investigate the functional underpinnings that may explain and link these structural and behavioral differences. We used two-photon imaging to determine whether SRGAP2C expression changed neuronal firing dynamics and found that it increased response reliability and selectivity to whisker inputs, thus improving accuracy of sensory coding. This improvement may help to explain why SRGAP2C mice performed better in a cortex-dependent task that actively relies on engagement of multiple cortical regions. Moreover, by using a humanized SRGAP2C mouse model, our results provide a small step towards better understanding how experimental studies can be interpreted for and translated to humans.

Background: Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts. Methods: In this study, we created myocyte-specific active HDAC4 transgenic mice to examine the functional role of active HDAC4 in mediating myocardial I/R injury. Ventricular function was determined in the isovolumetric heart, and infarct size was determined using tetrazolium chloride staining. Results: Myocyte-specific overexpressing activated HDAC4 in mice promoted myocardial I/R injury, as indicated by the increases in infarct size and reduction of ventricular functional recovery following I/R injury. Notably, active HDAC4 overexpression led to an increase in LC-3 and active caspase 3 and decrease in SOD-1 in myocardium. Delivery of chemical HDAC inhibitor attenuated the detrimental effects of active HDAC4 on I/R injury, revealing the pivotal role of active HDAC4 in response to myocardial I/R injury. Conclusions: Taken together, these findings are the first to define that activated HDAC4 as a crucial regulator for myocardial ischemia and reperfusion injury.

We investigated the effects of dietary α-tocopherol and polyunsaturated fatty acids (PUFA) on ischemia-reperfusion injury and cardiac lipid composition. Rats were fed corn oil (CO) diets either deficient (CO - E) or supplemented (CO + E) with RRR-α-tocopherol (100 IU kg-1 diet), or butter oil (BO) diets either deficient (BO - E) or supplemented (BO + E) with RRR-α-tocopherol (100 IU kg-1 diet). Intact rat hearts were subjected to ischemia before reperfusion. Dietary RRR-α-tocopherol supplementation contributed to recovery of aortic output, cardiac output and diastolic pressure after ischemia-reperfusion. In contrast, the type of dietary fat did not influence most measures of cardiac recovery. RRR-α-tocopherol levels in cardiac tissues and plasma were significantly higher for rats fed the BO + E diet than for rats fed the CO + E diet. In contrast to plasma, PUFA in cardiac tissues were maintained at a high level even when rats were fed BO containing diets. Our results suggest that dietary RRR-α-tocopherol, but not dietary PUFA levels, modulate oxidative damage to intact rat hearts during ischemia-reperfusion.

A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal α-sarcoglycan, γ-sarcoglycan, αDG, or βDG was observed. The maintenance of sarcolemmal βDG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of βDG. However, during late in vitro ischemia, a 30 Kd band was observed that was immuno-reactive for βDG. Additionally, this 30 Kd-βDG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-βDG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120-180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, βDG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-βDG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-βDG might mediate the cardioprotection provided by ischemic preconditioning.

No description available.

We describe the case of a patient with documented temporal arteritis, who presented two years into her course with acute digital ischemia, presumed secondary to small vessel vasculitis. To our knowledge, this complication of temporal arteritis has not been previously reported.

Background: Erythropoietin (EPO) can reduce myocardial ischemia/reperfusion (I/R) injury. However, the cellular mechanisms have not been elucidated entirely. The present study was to investigate whether transcription factor GATA-4 could be involved in EPO-induced cardioprotection when it was administered after ischemia, immediately before reperfusion. Methods and results: Male Balb/c mice treated with or without EPO were subjected to ischemia (45 min) followed by reperfusion (4 h). TTC staining showed that the infarct size in EPO-treated mice was significantly reduced compared with untreated I/R mice (P < 0.05). Echocardiography examination suggested that EPO administration significantly improved cardiac function following I/R. TUNEL assay indicated that EPO treatment decreased apoptosis. EPO administration also significantly increased the level of nuclear GATA-4 phosphorylation in the myocardium which was positively correlated with the reduction of myocardial infarction. In vitro hypoxia/re-oxygenation study showed that EPO treatment increased the levels of phospho-GATA-4 and decreased cardiomyocyte apoptosis. More significantly, blocking GATA-4 by transfection of a dominant-negative form of GATA-4 (dnGATA-4) abolished EPO-induced cardioprotective effects. Conclusion: EPO administration after ischemia, just before reperfusion induced cardioprotection and stimulated GATA-4 phosphorylation. Activation of GATA-4 may be one of the mechanisms by which EPO induced protection against myocardial I/R injury.

Inhibitors of apoptosis proteins (IAPs) are key intrinsic regulators of caspases-3 and -7. During ischemia, IAP-2 is upregulated dramatically, while the other IAPs show little or no change. To test whether IAP-2 prevents cardiac apoptosis and injury following ischemia/reperfusion, we generated a line of transgenic mice that carried a mouse IAP-2 transgene. High levels of mouse IAP-2 transcripts and 70 kDa IAP-2 were expressed in the hearts of transgenic mice, whereas IAP-1 and XIAP levels remained the same. Immunohistochemical studies revealed more intense staining of IAP-2 in the myocytes of transgenic mouse hearts. To assess the role of IAP-2 in I/R injury, the transgenic mice were subjected to ligation of the left descending anterior coronary artery ligation followed by reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (30 ± 2% vs. 44 ± 2%, respectively, P < 0.05). This protection was accompanied by a decrease of the serum level of troponin I in the transgenic mice. IAP-2 transgenic hearts had significantly fewer TUNEL-positive cardiac cells, which indicated an attenuation of apoptosis. Our results demonstrate that overexpression of IAP-2 renders the heart more resistant to apoptosis and I/R injury.

BACKGROUND AND PURPOSE - Humanin (HN) is a 24-amino acid peptide best known for its ability to protect neurons from damage caused by Alzheimer disease-related proteins. This study examines the neuroprotective effects of HNG (a potent form of HN) on focal cerebral ischemia/reperfusion injury in mice. METHODS - Mice underwent middle cerebral artery occlusion for 75 minutes followed by 24-hour reperfusion. Mice were pretreated with 0.1 μg HNG (intracerebroventricularly) 30 minutes before ischemia; posttreated at 0, 2, 4, and 6 hours after ischemia; or pretreated with 1 μg HNG (intraperitoneally) 1 hour before ischemia. Neurological deficits and cerebral infarct volume were evaluated. Neuronal apoptosis and activated poly(ADP-ribose) polymerase expression were measured by TUNEL and Western blot analysis, respectively. Activated ERKs were examined by Western blot analysis. RESULTS - Pretreatment with 0.1 μg HNG (intracerebroventricularly) 30 minutes before ischemia reduced cerebral infarct volume from 56.2±3.0% to 26.1±1.4% (P<0.01). HNG posttreatment after 4 hours of reperfusion reduced cerebral infarct volume to 45.6±2.6% (P<0.05). Pretreatment with 1 μg HNG (intraperitoneally) 1 hour before ischemia or posttreatment after 2 hours of reperfusion reduced cerebral infarct volume significantly. HNG also significantly improved neurological function and inhibited both neuronal apoptosis as well as poly(ADP-ribose) polymerase activation. A significant decrease of phospho-ERK was observed in mice treated with HNG, whereas phospho-JNK and phospho-p38 levels were not altered. CONCLUSIONS - Our results demonstrate that HNG protects against cerebral ischemia/reperfusion injury in mice. HNG offers neuroprotection in vivo at least in part by inhibiting ERK activation. These findings suggest a potential therapeutic role for HNG in the treatment of stroke.