Introdução: A torção testicular permanece como uma emergência urológica, despertando grande interesse em fármacos que podem minorar a lesão testicular e suas repercussões na fertilidade e produção hormonal. No entanto, não há fármaco aprovado para uso clínico rotineiro. Uma droga estudada em isquemia celular é a clorpromazina, sendo conhecidos seus efeitos protetores na função e estrutura da membrana celular e mitocondrial. Objetivos: Avaliar a diferença na lesão de células germinativas após 1 e 6 horas de torção e a ação da clorpromazina administrada previamente à resolução da torção no testículo isquêmico. Materiais e Métodos: 54 ratos Wistar, machos, com peso corporal entre 220 e 260 gramas distribuídos em 5 grupos: sham, controle com isquemia de 1 hora(A), controle com isquemia de 6 horas(B), experimental com isquemia de 1 hora(C) e experimental com isquemia de 6 horas(D). Em 48 animais foi realizada torção unilateral do cordão espermático com duas voltas em torno do seu eixo (720 graus), fixando-se o testículo nessa posição, após o que cada subgrupo foi separado em avaliação imediata (orquiectomia bilateral ao final do período de torção = 1) e tardia (orquiectomia bilateral, uma semana após a resolução da torção = 2). O grupo experimental recebeu 3 mg/kg de clorpromazina administrada via endovenosa, 30 minutos antes da resolução da torção. O grupo controle recebeu apenas solução salina a 0,9% por via endovenosa. Outros 6 animais formaram o grupo sham, onde foi realizada apenas a manipulação do cordão espermático. Após retiradas as gônadas, foram preparadas para análise histológica pela microscopia de luz e imunohistoquímica.Um pequeno fragmento de cada testículo foi separado para avaliação por microscopia eletrônica de transmissão (MET). Resultados: Na análise por microscopia de luz foram notadas alterações devido à isquemia como, necrose de coagulação e edema intersticial, principalmente nos grupos com isquemia mais prolongada (6h - B e D). Na avaliação por imunohistoquímica, houve maior expressão da caspase-3 nas células e túbulos dos testículos com 6 horas de isquemia, quando comparados com o grupo sham. No entanto, a expressão de bcl-2 não foi expressiva em nenhum grupo. Os grupos B e D também demonstraram alterações mais expressivas na análise por MET. Em nenhuma das avaliações foi observado superioridade do grupo da clorpromazina em relação ao grupo controle. Conclusão: As lesões celulares intratubulares induzidas pela isquemia e reperfusão testicular foram semelhantes após 1 e 6 horas, as diferenças foram relacionadas à sua maior intensidade no grupo com 6 horas e a clorpromazina não foi efetiva na prevenção da lesão por reperfusão. / Introdution: Testicular torsion remains as a urology emergency arousing interest about medicine which can reduce testicular injury and its impact on fertility and hormone production. However, there is no drug approved for routine clinical use. A drug studied in cell ischemia is chlorpromazine, being known its protective effects on the function and structure of cellular membrane and mitochondrial. Objective: To evaluate the difference in lesion of germ cells after 1 and 6 hours and the action of chlorpromazine administered before the resolution of ischemic testicle due torsion. Materials and methods: 54 male Wistar rats weighing between 220 to 260 grams divided into five groups: sham, control with one hour of ischemia (A) control with six hours of ischemia (B) experimental with one hour of ischemia (C) and experimental six hours of ischemia (D). In 48 animals was performed unilateral torsion of the spermatic cord with two laps around its axis (720 degrees), keeping the testicle in this position. After that, each subgroup was divided into immediate evaluation (bilateral orchiectomy at end of the torsion period = 1) or later (bilateral orchiectomy after one week of torsion resolution = 2). The experimental group received 3 mg / kg chlorpromazine administered intravenously 30 minutes before the resolution of torsion. The control group received only saline 0.9% intravenously. Other 6 animals were in the sham group, which was held just handling the spermatic cord. After withdrawal, the gonads were prepared for histological analysis by light microscopy and immunohistochemistry. A small piece of each testis was separated for evaluation by electron microscopy. Results: In analysis by light microscopy, ischemic changes were rated as coagulative necrosis and interstitial edema mainly in groups with prolonged ischemia (6h - B and D). When analyzed by immunohistochemistry, there was greater expression of caspase-3 in cells and tubules of the testes with 6 hour of ischemia compared to the sham group. However, bcl-2 expression was not impressive in either group. B and D groups also showed more significant changes in the analysis by electron microscopy. None of the ratings has been shown superiority of chlorpromazine group over the control group. Conclusion: The germ cell damage induced by ischemia and reperfusion was similar after 1 and 6 hours, the differences were related to its greatest intensity in the group with 6 hours and chlorpromazine was not effective in preventing reperfusion injury.

Introduction: La lésion d'ischémie - reperfusion (I/R) intestinale est associée à une mortalité élevée.Le rôle de la transmigration des neutrophiles est probablement essentiel dans le dysfonctionnement de la barrière induit par la lésion I/R et la nécrose subséquente. Le but de notre étude était d'évaluer, au niveau pré-clinique, la valeur thérapeutique potentielle du peptide P8RI (un agoniste de CD31) dans la lésion I/R de l'intestine grêle.Méthodes: I/R intestinale a été induite chez des rats Wistar par clampage de l'artère mésentérique supérieure (AMS) pendant 30 min suivi par 4 h de reperfusion. Trois groupes de rats ont été comparés:P8RI (n = 20, I/R et P8RI 2,5 mg/kg/h de reperfusion), I/R témoins positifs (n = 21, I/R et infusion de sérum physiologique) et témoins (n = 14, dissection de l'AMS sans clampage et infusion de sérum physiologique). La matrix métallopeptidase 9 (MMP-9) dans la plasma, le liquide péritonéal et l'intestin; la MPO intestinale, l'ADN libre plasmatique, les taux d'hémoglobine intraluminale e tl'épaisseur épithéliale ont été évalués comme marqueurs intermédiaires de la lésion intestinale. La détection du matériel génétique d'E.coli par PCR quantitative dans le sang a été également réalisée chez les témoins positifs et chez les rats traités par P8RI pendant la période de reperfusion. Le CD31clivé dans le plasma a été mesuré par une méthode d'ELISA maison. Résultats: La grade histologique de l'intestin grêle chez les animaux traités par P8RI est supérieur àcelui des témoins positifs d'I/R (p < 0,05). P8RI protège l'intestin grêle contre la destruction épithéliale induite par lésion I/R (p < 0,01). Il y a une corrélation négative significative entre l'abrasion épithéliale et le score de Chiu de classement histologique de la lésion I/R mésentérique (r = -0,7245, p < 0.001).P8RI protège l'intestin grêle de la MPO in situ, de la libération de la MMP-9 (p < 0,05) et des complications hémorragiques digestives causées par lésion I/R (p < 0,01). Les taux plasmatiques et péritonéaux accrus de MMP-9, induits par I/R, sont significativement réduits par P8RI (p < 0,05 et p <0,01, respectivement). L'ADN d'E.coli circulant est significativement plus élevé chez les rats de témoins positifs que dans le groupe P8RI (p < 0,05). L'ADN libre plasmatique pendant la reperfusion chez les rats de témoins positifs augmente significativement plus que celle des rats du groupe P8RI (p< 0,05). P8RI n'a aucun effet sur le compte neutrophilaire sanguin, mais induit une diminution significative de CD31 clivé dans le plasma pendant l'I/R mésentérique (p < 0,01). Les corrélations entre l'abrasion épithéliale et l'ADN libre, MMP-9, CD31 clivé plasmatiques, les taux de MMP-9 et MPO intestinale suggèrent que le mécanisme protecteur de P8RI implique l'inhibition de l'activation neutrophilaire, y compris le clivage de CD31. Conclusions: Cette étude suggère que le traitement par le peptide P8RI, un agoniste de CD31, réduit de la façon préventive, la lésion I/R de l'intestin grêle chez les rats. / Background: Intestinal ischemia/reperfusion (I/R) injury is associated with a high mortality. The roleof transmigration of PMNs is probably essential in I/R-induced intestinal barrier dysfunction andsubsequent necrosis. The aim of this study was to evaluate, at a preclinical level, the potentialtherapeutic value of P8RI peptide (a CD31 agonist), in small bowel I/R injury.Methods: Intestinal I/R was induced in Wistar rats by superior mesenteric artery (SMA) clamping for30 min followed by 4 h of reperfusion. Three groups of rats were compared: P8RI (n=20, I/R and P8RI2.5mg/kg/h infusion), I/R positive controls (n=21, I/R and normal saline infusion), and sham operatednegative controls (n=14, SMA dissection with no clamping and normal saline infusion). Plasma,peritoneal fluid and intestinal matrix metallopeptidase 9 (MMP-9), intestinal MPO, plasma cf-DNA,intraluminal hemoglobin levels and epithelial thickness were all evaluated as intermediate markers ofintestinal injury. Detection of E.coli genetic material by quantitative PCR in blood was also performedin I/R controls and P8RI-treated rats during the reperfusion period. Plasma cleaved CD31 wasmeasured by homemade ELISA.Results: The small bowel histologic grade in P8RI-treated animals was higher than in I/R controls (p <0.05). P8RI protected against epithelial destruction induced by I/R injury (p < 0.01). There was asignificant negative correlation between epithelial abrasion and Chiu's score of histologic grading ofmesenteric I/R injury (r = -0.7245, p < 0.001). P8RI protected the small bowel from in situ MPO andMMP-9 release (p < 0.05) and digestive bleeding complications due to I/R injury (p < 0.01). Theincreased plasma and peritoneal MMP-9 levels induced by I/R were significantly reduced by P8RI (p <0.05 and p < 0.01, respectively). Plasma cf-DNA during reperfusion in I/R controls increasedsignificantly more than in the P8RI group (p < 0.05). E.coli DNA was significantly higher in I/Rcontrols than in the P8RI group (p < 0.05). P8RI had no effect on blood neutrophil counts but induceda significant diminution of cleaved CD31 in plasma during mesenteric I/R (p < 0.01). Correlationsbetween epithelial abrasion and plasma cf-DNA, MMP-9, cleaved CD31, intestinal MMP-9 and MPOlevels suggest that the protective mechanism of P8RI might involve the inhibition of neutrophilactivation, including CD31 cleavage.Conclusions: This study suggests that a treatment by P8RI peptide, a CD31 agonist, reduces smallbowel I/R injury in rats

A long held hypothesis in mitochondrial biology holds that increases in mitochondrial Ca2+ levels stimulate the activity of matrix dehydrogenases that catalyze production of NADH and eventually donate electrons to electron transport in order to increase ATP formation. At the same time, mitochondrial Ca2+ overload is a deleterious event leading to opening of the mitochondrial permeability transition pore, increasing reactive oxygen species and initiating pathways that contribute to cell death. These fundamental hypotheses are best studied in the heart because of the critical energy supply-demand relationship in myocardium, but were untestable in vivo until the discovery of the mitochondrial Ca2+ uniporter (MCU). The molecular identity of the MCU pore forming subunit was recently discovered, which allowed me to study a transgenic mouse with myocardial delimited expression of a dominant negative MCU. My lab developed mice with myocardial-delimited transgenic expression of a dominant negative MCU to test these fundamental hypotheses and to determine how MCU controls physiological and pathological stress responses in vivo, ex vivo, and in situ. My studies provide new, unanticipated information that contributes to our understanding the relationship between mitochondrial Ca2+, oxygen utilization, cardiac pacemaking and pathologic stress responses in heart. Here, I show that mice with myocardial-targeted MCU inhibition have hearts with surprisingly high oxygen consumption rates due to elevated cytoplasmic Ca2+ in response to physiological stress. Loss of MCU effectively preserved inner mitochondrial membrane potential and prevented an oxidative burst thought to drive myocardial injury and death, but nevertheless failed to protect myocardium from ischemia-reperfusion injury. Increases in oxygen consumption, elevation in cytoplasmic Ca2+ and transcriptional reprogramming mitigate the protective actions of MCU inhibition in vivo. Mice with myocardial selective MCU inhibition have a reduced response to isoproterenol-induced heart rate increase but have normal baseline heart rates. My studies provide novel insight into how MCU contributes to myocardial Ca2+ homeostasis, metabolism, and transcription leading to surprising actions on physiological and pathophysiological responses in heart.

Matrix metalloproteinase2 (MMP2) was recently revealed to have targets and actions within the cardiac myocyte. In ischemia/reperfusion (I/R) injury, MMP2 is activated and degrades troponin I (TnI) and actinin. The regulation of intracellular MMP2 activity is relatively unknown and is thus the subject of this thesis. The localization of MMP2 in caveolae of endothelial cells suggests that caveolin1 (Cav1) may play a role in regulating MMP2. Whether Cav1 is responsible for regulating MMP2 in the heart is unknown. A Cav1 knockout mouse model was used to explore the role Cav1 may play in the regulation of MMP2 activity. The initial studies found that MMP2 and Cav1 were colocalized in cardiomyocytes and that MMP2 activity in Cav1/ hearts was markedly enhanced. Additionally, the caveolin scaffolding domain inhibited MMP2 activity in a concentrationdependent manner. To explore whether increased MMP2 in Cav1/ hearts translates to impaired cardiac function, Cav1+/+ and Cav1/ isolated working hearts were physiologically challenged with increasing increments of left atrial preload followed by increasing concentrations of isoproterenol. Cav1/ hearts show similar or better cardiac function compared to Cav1+/+ hearts following preload challenge or adrenergic stimulation in vitro, and this appears unrelated to changes in MMP2. Though the function of Cav1/ hearts appears similar to that of Cav1+/+ hearts during physiological situations, whether this is the case during I/R injury is not known. Cav1+/+ and Cav1/ isolated working mouse hearts exposed to global, noflow ischemia showed no functional differences. However, Cav1/ hearts had significantly higher levels of both TnI and actinin following I/R than Cav1+/+ hearts. Posttranslational modifications of the intracellular MMP2 substrates could alter susceptibility to MMP2 proteolysis. Isolated working mouse hearts were exposed to isoproterenol and/or I/R injury to examine the phosphorylation status of TnI. Isoproterenol and I/R both result in the phosphorylation of TnI, however, isoproterenol lead to a more highly phosphorylated form of TnI than that observed in hearts exposed I/R alone. These and subsequent studies will further reveal the molecular mechanisms that underlie the complex interactions between Cav1 and MMP2. This may eventually lead to a novel avenue of therapeutic intervention for heart diseases.

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.

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.

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.

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