京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21627号 / 医博第4433号 / 新制||医||1033(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 平井 豊博, 教授 松原 和夫, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

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.

No description available.

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

Liver transplantation is the only life-saving treatment for patients with end-stage liver disease; however, organ availability is insufficient to meet demands. Steatotic livers are extended criteria donor (ECD) organs that could be used for transplantation if not for an increased susceptibility ischemia reperfusion injury (IRI). Heme oxygenase-1 is a gene, that when upregulated has be shown to reduce IRI in animal models of transplantation. Increasing HO-1 activity in steatotic livers by delivery of a functional cell-penetrating HO-1 protein (through the use of cell-penetrating peptides) may provide protection against IRI, making these organs useful for transplantation. The purpose of this thesis was the generation and testing of a cell-penetrating HO-1 protein. HO-1 and EGFP gene sequences were cloned into the pET-28B(+) vector in frame with a CPP or TAT sequence. Resulting plasmids were cloned into E. coli, and protein expression was induced using IPTG. Proteins were purified using Ni-NTA affinity chromatography under denaturing and non-denaturing conditions. Non-denatured proteins were tested for HO-1 activity and the ability of both denatured and non-denatured proteins to transduce cells in vitro was tested by fluorescence microscopy. The cell-penetrating ability of nondenatured proteins was further tested in J774, HepG2 and HUVEC cells using immunofluorescence. Five HO-1 and two EGFP cell-penetrating proteins were generated expressed and purified successfully. Purified non-denatured HO-1 retains its enzymatic activity. Non-denatured CPP-EGFP and CPP-HO1 penetrated cells more effectively than their denatured counterparts. CPP-EGFP and CPP-HO1 proteins are able to penetrate multiple cell types in vitro. Successful generation and testing of a cell-penetrating HO-1 protein, for use in an animal model of steatotic liver transplantation. This protein demonstrates promise for use as a potential therapeutic agent in the field of liver transplantation.

The purpose of this study was to determine if del Nido cardioplegia provides superior protection for aged and young adult hearts. We used our isolated working heart model of cardioplegic arrest and reperfusion to compare functional recovery in both senescent and young adult rat hearts, with delivery of del Nido or our standard cardioplegia. In the aged hearts, use of del Nido cardioplegia prevented spontaneous contractions during arrest, reduced troponin release, and provided superior functional recovery during working heart. In contrast, in the young adult hearts, although stroke work was higher in the del Nido group, there were no significant differences in spontaneous activity, troponin release, and cardiac output between del Nido and standard cardioplegia, suggesting that del Nido cardioplegia did not provide superior functional recovery in the young adult heart. Del Nido cardioplegia has the potential to provide superior myocardial protection for elderly patients undergoing cardiac surgery.

CapZ is an actin capping protein that locates at cardiac Z-discs and anchors sarcomeric actin [1]. Transgenic (TG) mice overexpressing CapZ in cardiac myocytes develop a lethal cardiac hypertrophy [2], while a large reduction in CapZ protein causes severe myofibrillar disarray and death [2]. However, a TG model that contains a modest reduction in cardiac CapZ protein levels is viable and is associated with decreased PKC-dependent regulation of myofilament function [3]. Given the well known role of PKC in myocardial pathogenesis, the general aim of this thesis was to investigate how the modest reduction in CapZ protein affects cardiac function in models of cardiac stress. I found that PKC-translocation to cardiac myofilaments during cold cardioplegic arrest impairs myofilament activation, and that decreased cardiac CapZ protein disrupts this pathway and provides cardioprotective benefit. Using an in vivo model of ischemia-reperfusion (IR), I made the novel discovery that myofilament-associated PKC is altered during prolonged global ischemia, and found that a CapZ deficiency affects the translocation of PKC to myofilaments in a time-dependent manner. Furthermore, I found that TG mice deficient in CapZ demonstrate significant reductions in IR injury, while providing enhanced cardioprotection following ischemic preconditioning. The cardioprotected phenotype of CapZ-deficient TG mice is associated with altered translocation of several PKC-isoforms to cardiac myofilaments. Finally, having uncovered new information about the activation of protein phosphatase type 2A (PP2A) in IR, I investigated the role of CapZ in PP2A-dependent myofilament regulation. I found that reductions in CapZ may affect cardiac contractility by interrupting the association of PP2A with myofilaments. Together these findings expand the role of CapZ as a regulator of intracellular signaling molecules and demonstrate the novel ability of reduced CapZ to protect the heart against significant pathological threats. / Canadian Institutes of Health Research (CIHR), Heart and Stroke Foundation of Ontario (HSFO), Heart and Stroke Foundation of Canada (HSFC), The Premier's Research Excellence Award (PREA), Ontario Graduate Scholarship Program (OGS).

Background/Objective: Reperfusion injury is a serious consequence of blood flow reestablishment after myocardial infarction (MI) mediated by reactive oxygen species and neutrophilic cellular damage. Following MI, the left ventricle (LV) undergoes remodeling characterized by progressive wall thinning and cavity dilatation. Heme-Oxygenase-1 (HO-1) dependent decrease in oxidative stress may attenuate injury in part by inhibiting transcription factor NFκB-mediated inflammation. Hypothesis: I hypothesized that upregulation of HO-1 by hemin administration confers acute and chronic cardioprotection against I/R injury in rats and attenuates LV remodeling post-MI. I proposed the HO-1-dependent decrease in oxidative stress attenuates post-ischemic myocardial injury in part by inhibiting NFκB-mediated inflammation. Methods: Six week old male Wistar rats were randomly assigned to sham, vehicle, or hemin-treated groups. Vehicle and hemin were administered intraperitoneally once daily for 3 consecutive days prior to left anterior descending (LAD) coronary artery occlusion. Administration resumed 48 hours post-operatively and continued once every 3 days. Infarct size was determined by H&E histological analysis and fibrosis was quantified by Masson’s Trichrome staining. Transthoracic echocardiography was used to assess LV parameters and wall motion. Results: Hemin increased HO-1 expression, decreased infarct size and fibrosis, and attenuated LV remodeling in the short-term (4 days post-infarction). The decrease in infarct size and area of fibrosis in the hemin group was accompanied by a decrease in NFκB activity. No significant difference in infarct size and area of fibrosis between hemin and vehicle-treated groups was observed at 3 months. LV diameter and cardiac function did not differ significantly between the two groups at 3 months despite an attenuation of anterior wall thinning in the hemin group. Conclusion: HO-1 upregulation by hemin administration conferred acute cardioprotection and attenuated LV remodeling, possibly by inhibiting NFκB-mediated inflammation. However, chronic treatment with hemin did not prevent long-term post-infarction LV remodeling. It is possible that cardioprotection afforded by HO-1 upregulation is strong enough to curtail inflammation post-reperfusion and prevent LV remodeling acutely, but is not robust enough to protect the myocardium to the same degree in the long-term. Future research should focus on optimal HO-1 upregulation to attenuate long-term LV remodeling due to reperfusion injury. / Thesis (Master, Physiology) -- Queen's University, 2007-09-25 19:01:33.87