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 柿沼善弘氏の博士論文として提出された

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 家田信人氏の博士論文として提出された

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成24年2月29日 石黒久昌氏の博士論文として提出された

Abstract The first part of this research was focused on the relationship between injury severity and cell death mechanisms after spinal cord ischemia-reperfusion injury. The major blood supply to the thoracolumbar spinal cord comes from the segmental arteries originating from the thoracoabdominal aorta. Paraplegia cause by spinal cord ischemia is a devastating complication of thoracoabdominal aortic surgery. Previous studies indicated that ischemia-reperfusion injury of the central nervous system causes two distinct types of cell death, necrosis and apoptosis. It was also implicated that the intensity of injury can somehow affect the cell death mechanisms. In the first series of our experiments, by occluding the descending thoracic aorta with or without simultaneously inducing hypovolemic hypotension in rats, we established a model of experimental spinal cord ischemia-reperfusion (SCIR) in which the injury severity can be controlled. Recordings of carotid blood pressure (CBP) and spinal cord blood flow (SCBF) showed that aortic occlusion induced dramatic CBP elevation but SCBF drop in both the normotensive (NT) and hypotensive (HT) groups. However, the HT group demonstrated significantly lower SCBF during aortic occlusion, and much slower elevation of SCBF after reperfusion, and extremely poor neurological performance. Spinal cord lesions were characterized by infarction associated with extensive necrotic cell death, but little apoptosis and caspase-3 activity. In contrast, in the NT group, SCIR resulted in minor tissue destruction associated with persistently abundant apoptosis, augmented caspase-3 activity, and favorable functional outcome. The relative sparing of motoneurons in the ventral horns from apoptosis might have accounted for the minor functional impairment in the NT group. The severity of ischemia-reperfusion (I/R) injury was found to have substantial impact on the histopathological changes and cell death mechanisms, which correlated with neurological performance. These findings implicate that injury severity and duration after injury are two critical factors to be considered in therapeutic intervention. Based on the knowledge that bPrevious studies have implicated both excitotoxicity and apoptosis are involved in the pathogenesis of SCIR injury, we proposedtested the possibility that the N-methyl-D-aspartate (NMDA) receptor antagonist (dizocilpine maleate: (MK801) and the protein synthesis inhibitor (cycloheximide) would produce a synergic effect in the treatment of SCIR injury. In the second series of experiments, I/R iSpinal cord ischemia-reperfusion injury was induced by a thoracic aortic occlusion and blood volume reduction, followed by reperfusion and volume restoration. ischemia-reperfusion Rats were treated with vehicle, MK801, cycloheximide, or combination of MK801 and cycloheximide in combination. The MK801 and combined therapy group got a better recovery of hHind limb motor function recovery was better in the MK801 and combined-therapy groups than in the control and cycloheximide groups. On the 7th day after ischemia-reperfusion injury, all three treated groups showed significantly higher neuronal survival rates (NSR) than that of the control group. Among the three treated groups, the combined-treatment group showed the highest NSR. In addition, the Ttherapeutic effect of the combined-treatment group (27.4% increase of NSR) iwas better than the anticipated by the addition of MK801 and cycloheximide based on NSR data group. The number of apoptotic cells of was significantly reduced in the cycloheximide group and the combined-treatment group, as compared to that of the control group. It was unchanged in the MK-801 group. These results suggest that combined treatments directed at blocking both NMDA receptor-mediated excitotoxic necrosis and caspase-mediated apoptosis might have synergic therapeutic potential in reducing SCIR injury. Mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal kinases (JNK), p38, and extracellular signal-regulated kinases (ERK), play important roles in the transduction of stressful signals and the integration of cellular responses. Although it has been generally held that the JNK and p38 pathways are related to cell death and degeneration, while the ERK pathway, cell proliferation and survival, controversy still exists. The roles of the ERK pathway in I/R injury of the CNS, in particular, remain to be clarified, because contradictory data have been reported by different investigators. Given this controversy, in the third series of experiments, we examined in injured spinal cords the temporal and spatial profiles of ERK1/2 activation following SCIR, and the effects of inhibiting the kinase that phosphorylates ERK1/2, MEK. The results showed that I/R injury induced an immediate phosphorylation of ERK1/2 in the spinal cord, which was alleviated by a MEK inhibitor, U0126. The control group was characterized by poorer neurological outcome, more severe tissue destruction, pronounced apoptosis, and lower neuronal survival. In contrast, the U0126-treated group demonstrated more apparent improvement of hind limb motor function, less tissue destruction, lack of apoptosis, and higher neuronal survival. In addition, administration of U0126 also significantly increased the activation of nuclear factor-£eB (NF-£eB) and the expression of cellular inhibitor of apoptosis protein 2 (c-IAP2). These findings implicate that the mechanisms underlying the neuroprotection afforded by ERK1/2 inhibition may be through the NF-£eB-c-IAP2 axis. The activation of the MEK-ERK signaling pathway appeared to be harmful in SCIR injury. Strategies aimed at blocking this pathway may bear potential therapeutic benefits in the treatment of SCIR injury. The second part of the research was focused on the pathophysiology of surgery-associated paraspinal muscle injury and measures to protect surgically violated paraspinal muscles. The wide dissection and forceful retraction of paraspinal muscles which are often required for posterior spinal sugery may severely jeopardize the muscles structurally and functionally. Immediate posteoperative pathological changes in the surgically violated paraspinal muscles may cause severe pain and a delay of patient ambulation. Long-term sequelae of surgical injury of paraspinal muscles include chronic back pain and impaired back muscle strength. Ironically, being a common complication of posterior spinal surgery, paraspinal muscle injury is so often neglected. Limited previous data indicate that the underlying pathophysiology of muscle damage involve both mechanical and ischemic mechanisms. We hypothesized that surgical dissection and retraction may produce oxidative stress within the paraspinal muscles. Meanwhile, we also proposed that the oxidative stress may trigger certain protective mechanisms within the insulted muscles. The first part of our study was a human study conducted to assess the significance of oxidative stress, and the relationship between it and the stress response mediated by heat shock protein 70 (HSP70) induction within paraspinal muscles under intraoperative retraction. A group of patients with lumbar spondylolisthesis treated with posterolateral lumbar spinal fusion, pedicle fixation and laminectomy were enrolled. Multifidus muscle specimens were harvested intraoperatively before, at designated time points during, and after surgical retraction. Muscle samples were analyzed for HSP70 and malondialdehyde (MDA) levels. Both HSP70 expression and MDA production within multifidus muscle cells were increased significantly by retraction. HSP70 expression then dropped after a peak at 1.5 hr of retraction, whereas MDA levels remained elevated even after release of retractors for reperfusion of the muscles. Histopathological and immunohistochemical evidence indicated that the decline of HSP70 synthesis within muscle cells after prolonged retraction was the result of severe muscle damage. These results highlighted the noxious impact of intraoperative retraction on human paraspinal muscles, and the significance of oxidative stress at the cellular and molecular levels. It is also implicated that intraoperative maneuvers aimed at reducing the oxidative stress within the paraspinal muscles may help attenuating surgery-associated paraspinal muscle damage. Given the findings of the first part of our study, and the knowledge that inflammation is a major postoperative pathological finding in surgically injured paraspinal muscles, we proceeded to examine the roles of two important inflammatory mediators, cyclooxygenase (COX)-2 and nuclear factor (NF)-£eB, in the pathogenesis of retraction-associated paraspinal muscle injury. A rat model of paraspinal muscle dissection and retraction that mimicks the conditions in human posterior spinal surgery was established. In the control group, paraspinal muscles were dissected from the spine through a dorsal incision, and then laterally retracted. Paraspinal muscle specimens were harvested before, and at designated time points during and after persistent retraction. The time course of NF-£eB activation as well as the expression of COX-2 were examined. Severity of inflammation was evaluated based on histopathology and myeloperoxidase (MPO) activity. NF-£eB activation was inhibited by the administration of pyrrolidine dithiolcarbamate (PDTC) in the PDTC-treated group. In the control group, retraction induced an early increase of NF-£eB/DNA binding activity in paraspinal muscle cells, which persited throughout the whole course of retraction. COX-2 expression was not detectable until 1 day after surgery, and reached a peak at 3 days. The time course of COX-2 expression correlated with that of inflammatory pathology and MPO activity. Extensive muscle fiber loss and collagen fiber replacement were observed at 7 days after surgery. Pretreatment with PDTC inhibited intraoperative NF-£eB activation and greatly downregulated postoperative COX-2 expression and inflammation in the muscles. Fibrosis following inflammation was also significantly abolished by PDTC administration. These findings indicate that NF-£eB-regulated COX-2 expression and inflammation play an important role in the pathogenesis of surgery-associated paraspinal muscle injury. Therapeutic strategies involving NF-£eB inhibition may be applicable to the prevention of such injury.

Mental stress ischemia (MSI) is associated with poor prognosis for coronary artery disease (CAD) and is amenable to treatment, yet no easily administered test exists to diagnose it. Given the known increase in systemic vascular tone in response to stress, we studied the ability of peripheral arterial tonometry (PAT), a noninvasive functional measure of arterial tone, to predict those vulnerable to MSI. Seventy-seven patients with chronic stable CAD were subjected to mental stress with concomitant assessment of myocardial perfusion and pulse wave amplitude. Nuclear perfusion imaging was used to document MSI, and PAT was used to measure pulse wave and microarterial tone. A ratio of PAT measurements during stress to those before stress was used to characterize vascular responses. Serum catecholamines and endothelin-1 (ET-1) were simultaneously measured. Subjects who experienced MSI had a lower average PAT ratio than those who did not (0.76 ¡À 0.04 vs. 0.91 ¡À 0.05, P = 0.03). A receiver operating characteristics curve for PAT ratio predicting MSI had an area under the curve of 0.613 (standard error, 0.065, one-sided P = 0.04). Maxima of sensitivity and specificity were observed at a threshold of 0.78 to define an abnormal PAT ratio. Cross-tabulation of groups above and below this threshold with groups of subjects with and without MSI showed a significant predictive relationship between PAT ratio and MSI (P = 0.03). Subjects at or below this threshold (¡Ü0.78) displayed a significant increase in norepinephrine levels during mental stress (235 pg/ml at baseline, 259 pg/ml during mental stress, P = 0.007). Subjects above this threshold (>0.78) displayed a significant decline in their ET-1 levels 24 hours after mental stress (1.15 pg/ml after mental stress, 0.93 pg/ml 24 hours later, P = 0.01), while those at or below threshold had a continued increase. PAT ratio is a complex functional measure of peripheral arterial tone that significantly predicts the occurrence of MSI. It may have clinical value as an easily administered screening test for MSI.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Physiology and Integrative Biology." Includes bibliographical references (p. 95-114).

Thesis (MD (Anaesthesiology))--University of Pretoria, 2003. / Includes bibliographical references.

Thesis (Ph. D.)--University of California, San Diego, 2003. / Vita. Includes bibliographical references (leaves 162-178).

Hepatic ischaemia-reperfusion injury occurs when patients undergoing liver operations such as liver transplantation, tumour resection and shock. Intravenous and intrathecal administration of morphine can be used to provide analgesia prior or after liver surgery. It has been reported that systemically administered morphine conferred protective effect on numerous organs, including heart, brain and kidney. The focus of my research is to investigate the effect of intravenous and intrathecal morphine preconditioning on normal and cirrhotic livers. Further, PI3K/Akt, STAT3 and HO-1/iNOS pathways had been shown to ameliorate hepatic ischemia-reperfusion injury. Hence, we aim to investigate these possible signaling pathways associated with morphine mediated hepato-protection. A partial hepatic ischaemia reperfusion injury model in rats was used. The experiments were divided into two series: one involved in normal livers and the other one involved in cirrhotic livers. For the normal livers, morphine at different doses were administrated intravenously or intrathecally prior the onset of ischaemia, and the experiments were repeated with previous intravenous administration of naloxone methiodide (opioid receptor antagonist), or wortmannin (Akt inhibitor), respectively. For the cirrhotic livers, morphine at optimal doses were injected intravenously or intrathecally prior the onset of ischaemia. Those rats with only induced hepatic ischaemia-reperfusion injury only were marked as control groups. The effect of morphine preconditioning on hepatic architecture, apoptosis and liver function were evaluated respectively by hematoxylin-eosin (H&E) staining, Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) staining, the expression of cleaved Caspase-3, and serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Meanwhile, the expression of phosphorylated Akt, phosphorylated JAK2, phosphorylated STAT3, HO-1 and iNOS were detected by Western Blot to determine the signaling pathways involved by intravenous and intrathecal morphine preconditioning. The normal livers series presented intravenous and intrathecal morphine preconditioning at the 100μg/kg, 10μg/kg, respectively, better persevered hepatic architecture when compared with control groups. The degree of liver cell apoptosis and expression of cleaved caspase-3 were also reduced by intravenous and intrathecal morphine preconditioning. In additional, intravenous and intrathecal morphine preconditioning ameliorated hepatocellular damage by reducing ALT&AST release. Moreover, the expressions of phosphorylated Akt and its downstream protein STAT3 were significantly increased by intravenous and intrathecal morphine preconditioning, compared with their respective control groups. The hepato-protective effect of intravenous and intrathecal morphine preconditioning was reversed by naloxone methiodide or wortmannin pretreatment. The similar pattern of protection was observed in cirrhotic livers. Both intravenous and intrathecal morphine preconditioning protected hepatic architecture much better than control groups. They also attenuated hepatic apoptosis degree and hepatocellular enzyme release. Furthermore, the expression of HO-1 was up-regulated, whereas the expression of iNOS was down-regulated by intravenous and intrathecal morphine preconditioning. In summary, this study provided evidence that intravenous and intrathecal morphine preconditioning could attenuate hepatic ischaemia-reperfusion injury in normal and cirrhotic livers. The involvement of opioid receptors, Akt/STAT3 pathway and HO-1 pathway might be the underlying mechanisms of morphine hepato-protection. Finally, the protective effect of morphine preconditioning might provide a potential therapeutic approach for clinical usage. / published_or_final_version / Anaesthesiology / Master / Master of Philosophy

Remifentanil is an ultra-short-acting phenylpiperidine opioid analgesic that is rapidly metabolized by nonspecific blood and tissue esterases. In clinical practice, remifentanil is now more commonly used during both cardiac and non-cardiac surgery than classic opioid agonists such as morphine, since it can be given in higher doses, is more titratable and enables fast recovery of patients in the postoperative period. Remifentanil preconditioning (RPC), achieved by intravenous remifentanil infusion interspersed with infusion-free periods before indexed ischaemia, attenuates cardiac ischaemia-reperfusion injury (IRI). This is experimentally manifested by reduced postischaemic myocardial infarct size (IS) and diminished markers of cardiac failure and apoptosis, and, clinically, by reduced release of biomarkers of myocardial cellular injury after cardiac surgery. However, the underlying mechanisms by which RPC has a cardioprotective effect need to be further explored. It’s generally considered that the Reperfusion Injury Salvage Kinase (RISK) pathway, triggering the expression of phosphatidylinositol 3-kinase (PI3K) as well as Akt, exerts a pivotal role in both classic ischaemic preconditioning (IPC) and pharmacological preconditioning induced cardioprotection. Moreover, recent studies show that the Survivor Activating Factor Enhancement (SAFE) signalling pathway, which involves signal transducers and activators of transcription-3 (STAT3) and janus activated kinase-2 (JAK2), also has an essential role in IPC. Although cross-talk has been found between the RISK and SAFE pathways, the SAFE pathway can function independently of the RISK to confer cardioprotection. However, the roles of JAK/STAT and PI3K/Akt signalling and, in particular, their relative importance in RPC-mediated cardioprotection have not been studied. I explored whether RPC confers cardioprotection via the JAK/STAT or PI3K/Akt pathway and its relationship with GSK3β inhibition. In first part of my study, I explored relative role of the JAK/STAT and PI3K/Akt which were involved in RPC cardioprotection using JAK2 and PI3K inhibition. Male Sprague-Dawley rats were either sham operated or randomly assigned to receive I/R alone or as well as RPC. Pretreatment with the JAK2 inhibitor AG490 or the PI3K inhibitor wortmannin was induced before ischaemia in rats. RPC reduced myocardial infarction and haemodynamic dysfunction induced by IRI accompanied with increased phosphorylation of STAT3 but not Akt or eNOS phosphorylation. AG490 but not wortmannin cancelled RPC’s cardioprotection. In addition, RPC attenuated hypoxia/reoxygenation induced cardiomyocyte apoptosis while STAT3 knock-out abolished the protective effects of RPC. These findings suggest that RPC confers cardioprotection primarily via activation of the JAK/STAT signalling but not the PI3K/Akt signalling pathway. The second study further investigated the role of GSK3β in RPC cardioprotection using the GSK3β inhibitor SB216763. I found that SB restored the ability of RPC to reduce the extent of myocardial infarction and CK-MB release despite the presence of AG490. The phosphorylation of GSK3β was increased by RPC. In addition, GSK3β gene knock-out with siRNA preserved RPC’s cardioprotection regardless of STAT3 abrogation indicating that GSK3β inhibition plays a critical role as a downstream effector in RPC mediated cardioprotection. Taken together with the evidence from this two part study, I conclude that RPC confers cardioprotection by activating the JAK/STAT and, subsequently, inhibiting GSK3β, a critical downstream effector of RPC cardioprotection. / published_or_final_version / Anaesthesiology / Master / Master of Philosophy