Literatura científica selecionada sobre o tema "Reperfusion injury Pathophysiology"

Prolonged ischaemia sometimes occurs in replantation and free flap surgery. The re-establishment of circulatory flow to the ischaemic tissue leads to a cascade of events which augments tissue necrosis. This paper reviews the pathophysiology of this ischaemia-reperfusion injury and discusses different methods to modulate this injury.

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p<0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p<0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p<0.001), leading to a 60% survival rate at 48 hours IRI (p<0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p<0.001), with a survival benefit of 75% (p<0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p>0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Includes bibliographical references (leaves 292-352) Determines the role of matrix metalloproteinases, MMP-2 and MMP-9 in reperfusion injury following skeletal muscle ischaemia; and, whether inhibition of MMPs by doxycycline protects against tissue damage.

Las alteraciones mitocondriales están vinculadas a la mayor vulnerabilidad de padecer enfermedades durante el envejecimiento. La edad avanzada es un factor determinante de la incidencia y gravedad de la cardiopatía isquémica. Estudios preclínicos sugieren la existencia de un daño celular intrínseco, por mecanismos no del todo establecidos, que contribuye a un incremento de la susceptibilidad del miocardio senescente al daño isquémico. Esta tesis investiga el papel de la comunicación mitocondria-retículo sarcoplásmico (RS) en el deterioro funcional de los cardiomiocitos durante el envejecimiento. El estudio ecocardiográfico ha demostrado que la función cardiaca en el reposo se mantiene preservada en los animales viejos. No se han observado cambios debidos a la edad en el potencial de membrana mitocondrial ni en el consumo de oxígeno en condiciones de reposo en mitocondrias aisladas de corazones de ratón. El consumo de oxígeno inducido por ADP ha revelado que las mitocondrias interfibrilares de corazones viejos no alcanzan el nivel respiratorio máximo. Análisis proteómicos de segunda generación han demostrado un aumento de la oxidación de proteínas mitocondriales relacionado con el envejecimiento. Esta tesis ha investigado el posible efecto de la edad sobre la capacidad de las mitocondrias para captar calcio. En cardiomiocitos la captación mitocondrial del calcio procedente del RS se ha visto reducida de forma significativa en el envejecimiento. Esta disminución de la captación de calcio mitocondrial se asoció a una reducida capacidad de regeneración de NAD(P)H y a un incremento de la producción de ROS mitocondriales en cardiomiocitos viejos. Ensayos de inmunofluorescencia y de ligación por proximidad han revelado una comunicación defectuosa entre la mitocondria y el RS en cardiomiocitos de corazones senescentes. La desestructuración de las uniones entre el RS y la mitocondria con colchicina fue capaz de reproducir el efecto de la edad sobre las alteraciones en el manejo/transferencia de calcio entre ambos orgánulos en cardiomiocitos jóvenes. La segunda parte de este trabajo investiga el impacto potencial de las alteraciones mitocondriales sobre los efectos adversos del envejecimiento en el daño por isquemia y reperfusión (IR). Los corazones aislados y perfundidos de ratones viejos sometidos a IR desarrollaron mayor rotura sarcolemal y tamaño de infarto, junto con un retraso significativo del desarrollo del rigor isquémico. Los cardiomiocitos viejos sometidos a isquemia, desarrollaron una caída más rápida del potencial de membrana mitocondrial (ΔΨm) junto con un retraso paradójico en la aparición del rigor. La tasa de recuperación transitoria del ΔΨm durante los primeros minutos de isquemia, debida a la actividad reversa de la FoF1-ATPsintasa, se encontró significativamente disminuida en cardiomiocitos viejos. El análisis proteómico ha demostrado un aumento de la oxidación de diferentes subunidades de la FoF1-ATPsintasa asociado al envejecimiento. La alteración del ΔΨm observado en los cardiomiocitos viejos se asoció a una menor captación de calcio mitocondrial durante la IR. A pesar de esto, el desarrollo de permeabilidad transitoria (mPT) fue mayor en los cardiomiocitos senescentes y este efecto se correlacionó con una mayor hipercontractura y muerte celular en reperfusión. Por lo tanto, el desarrollo de una comunicación defectuosa entre el RS y la mitocondria durante el envejecimiento produce un intercambio ineficiente de calcio entre ambos orgánulos, que contribuye al desajuste en la demanda/aporte de energía y a un aumento consiguiente del estrés oxidativo. La oxidación de la FoF1-ATPsintasa se asocia a una alteración de su funcionamiento y a un incremento de la sensibilidad de la mitocondria para desarrollar mPT. Debido al modelo recientemente propuesto según el cual la FoF1-ATPsintasa forma parte del mPTP, es posible especular que la oxidación de esta enzima está asociada al aumento del daño por IR en el miocardio senescente.
Mitochondrial alterations are critically involved in the increased vulnerability to disease during aging. On the other hand, aging is a major determinant of the incidence and severity of ischemic heart disease. Preclinical information suggests the existence of intrinsic cellular alterations that contribute to ischemic susceptibility in senescent myocardium, by mechanisms not well established. The first part of this thesis investigates the contribution of mitochondria-sarcoplasmic reticulum (SR) communication in the functional decline of cardiomyocyte during aging. Echochardiographic analysis of aging mice (>20 months) showed a rather preserved cardiac contractile function in resting conditions respect to young mice (5-6 months). ATP/phosphocreatine were preserved in hearts from old mice as quantified by RMN spectroscopy. In isolated mitochondria from young and old mouse hearts, mitochondrial membrane potential and resting O2 consumption were similar in both groups. However, stimulation of O2 consumption after the addition of ADP resulted in a partial failure of interfibrillar mitochondria from aged hearts to achieve maximal respiratory rate. Second generation proteomics disclosed an increase of mitochondrial protein oxidation in advanced age. Because both energy production and oxidative status are regulated by mitochondrial calcium, this work further investigated the effect of age on mitochondrial calcium uptake. While no age-dependent differences were found in calcium uptake kinetics in isolated mitochondria, in which the contribution of other organelles and sarcolemma is absent, mitochondrial calcium uptake secondary to SR calcium release was significantly reduced in cardiomyocytes from old hearts. Reduced mitochondrial calcium uptake in aging cardiomyocytes was associated with decreased NAD(P)H regeneration and a concomitant increase of mitochondrial ROS production manifested only when cells were exposed to high frequency electrical stimulation. Immunofluorescence and proximity ligation assay identified defective communication between mitochondria and SR in cardiomyocytes from aged hearts. Functional analysis of calcium handling in fluo-4 loaded cardiomyocytes disclosed an altered pattern of RyR gating properties. The observed defects in SR calcium transfer and in calcium handling could be reproduced in young cardiomyocytes after interorganelle disruption with colchicine, at concentrations that had no significant effect in aged cardiomyocytes or isolated mitochondria. The second part of this work investigates the potential impact of the altered mitochondrial function in the adverse effect of aging on myocardial ischemia and reperfusion (IR) injury. Isolated perfused hearts from old mice submitted to transient IR displayed an increase in hypercontracture, sarcolemmal rupture and infarct size, as compared to hearts from young mice, despite a paradoxical delay ischemic rigor contracture onset. In isolated cardiomyocytes from aging hearts submitted to IR there was a faster decline of mitochondrial membrane potential (ΔΨm) in comparison with young ones, but ischemic rigor shortening was also delayed. Transient recovery of ΔΨm observed during ischemia, secondary to the reversal of mitochondrial FoF1-ATPsynthase to ATPase mode, was markedly reduced in aging cardiomyocytes. Proteomic analysis demonstrated an increased oxidation of different subunits of FoF1-ATPsynthase. Altered bionergetics in aging cells was associated with reduced mitochondrial calcium uptake and more severe cytosolic calcium overload during both ischemia and reperfusion. Despite attenuated mitochondrial calcium overload, the occurrence of mitochondrial permeability transition pore (mPTP) opening, hypercontracture and cell death were increased during reperfusion in cardiomyocytes from old mice. In vitro studies demonstrated a significantly reduced calcium retention capacity in interfibrillar mitochondria from aging hearts. Thus, defective SR-mitochondria communication underlies inefficient interorganelle calcium exchange that contributes to energy demand/supply mismatch and oxidative stress in the aged heart. This may spread on an altered FoF1-ATPsynthase and increased sensitivity of mitochondria to undergo mPTP opening as important determinants of the reduced tolerance to ischemia-reperfusion in senescent myocardium. Because ATPsynthase has been proposed to conform mPTP, it is tempting to hypothesize that oxidation of ATPsynthase underlie both phenomena.

博士
國立臺灣大學
生理學研究所
92
Ischemia-reperfusion injury occurrs when the reoxygenated blood is introduced into anoxic tissue, and that cause multiple organs dysfunction. Our studies aim to understand the pathophysiology and to the way to minimize the ischemia-reperfusion injury. Recently, investigators found that heme oxygenase-1, a key enzyme in heme metabolism, was highly expressed when cells were experiencing stress, and the expression was supposed to be cell-protective. To clarify the mechanism of preconditioning, we used the liver ischemia-reperfusion model to testify the role that heme oxygenase-1 plays in two kinds of preconditioning, hypoxic and remote preconditioning. Besides, to understand the pathophysiology of ischemia-reperfusion injury on remote organs, we used the intestinal ischemia-reperfusion injury to testify the associated changes of renal nerve activity and renal function. In the first experiment, we proposed that hypoxic preconditioning (HP) confers cytoprotection against ischemia-reperfusion injury, and this effect is in part due to the induction of heme oxygenase-1. This experiment evaluates liver cell damage after ischemia-reperfusion injury in HP rats. HP rats were prepared by exposure (15hours day-1) to an altitude chamber (5500m) for 2 weeks. Partial hepatic ischemia was produced in the left lobes for 45 minutes followed by 180 minutes of reperfusion. Zinc-protoporphyrin IX(ZnPP), a specific inhibitor of HO enzymatic activity, was subcutaneously injected 1 hour before the I/R injury in separate groups of sea-level （SL）control and HP rat. Serum alanine transaminase (ALT) levels, liver HO-1 mRNA and protein, and HO enzymatic activity were measured. Our results showed that heme oxygenase-1 (HO-1) was induced in the livers of rats exposed to HP. The levels of HO-1 mRNA and protein were obviously over-expressed after two weeks of hypoxic preconditioning. HP diminished the elevation of serum ALT levels after I/R injury （83.7±4.9 U L-1）when compared with SL controls （280.8±19.4 U L-1） and HP+ ZnPP pre-treated groups（151.3±4.4 U L-1）. The heme oxygenase activity in treated rats also correlated these results（237.9±19.8 pmol mg-1 protein hr-1 for the HP group, 164.3±12.7 pmol mg-1 protein hr-1 for the HP+ ZnPP, and 182.6±8.9 pmol mg-1 protein hr-1 for the SL controls. Our data showed that (a）The induction of HO-1 in HP indicates that it may participate in the cellular response to hypoxia；（b） hypoxic preconditioning protects the liver from ischemia-reperfusion injury；（c） the protective effects induced by hypoxic preconditioning are reduced by inhibiting HO-1 enzyme activity with pretreated ZnPP, suggesting that the effects are mediated by HO-1. In the next experiment, again we proposed that remote preconditioning (RP) confers cytoprotection against ischemia-reperfusion injury, and this effect is in part due to the induction of heme oxygenase-1. This experiment evaluates liver cell damage after ischemia-reperfusion injury in RP rats. The remote preconditioning was produced by four cycles of 10 minutes’ ischemia-reperfusion of the hind limb of rats. Partial hepatic ischemia was produced in the left lobes for 45 minutes followed by 180 minutes of reperfusion. Zinc-protoporphyrin IX(ZnPP), a specific inhibitor of HO enzymatic activity, was subcutaneously injected 1 hour before the ischemia-reperfusion injury in separate groups of control and RP rat. Serum alanine transaminase (ALT) levels, liver HO-1 protein, and HO enzymatic activity were measured. Our results showed that heme oxygenase-1 (HO-1) was induced in the livers of rats exposed to RP (2793.6± 422.7 V.S. 1614.7±454.2 unit). RP diminished the elevation of serum ALT levels after I/R injury （346.5± 251.4 U L-1）when compared with controls （1188.3±559U L-1） and RP+ ZnPP pre-treated groups（1578± 692.3U L-1）. The heme oxygenase activity in treated rats also correlated these results（286.8±34.3 pmol mg-1 protein hr-1 for the RP group, 156.3±27.5 pmol mg-1 protein hr-1 for the RP+ ZnPP pre-treated group, and 170.6±19.4pmol mg-1 protein hr-1 for the control group, 144.8± 7.8pmol mg-1 protein hr-1 for the control+ ZnPP pre-treated group）. Our data showed that (a）remote preconditioning produced by repeated limb ischemia-reperfusion could induce hepatic HO-1 expression；（b） remote preconditioning protects the liver from ischemia-reperfusion injury；（c） the protective effects induced by remote preconditioning are reduced by inhibiting HO-1 enzyme activity with pretreated ZnPP, suggesting that the effects are mediated by HO-1. The above two in vivo experiments showed that HO-1 plays important roles in both the mechanisms of hypoxic and remote preconditioning. Previous study showed that a neurogenic pathway was involved in the mechanism of remote intestinal preconditioning. We proposed that remote organs injury induced by the intestinal reperfusion injury might be related to neurogenic mechanisms. To clarify this problem, we used the intestinal ischemia-reperfusion injury model to testify the impact of the injury upon renal nerve activity and associated renal dysfunction. Our results showed that the efferent renal nerve activity (ERNA) was only 14.3±6.6% lower than basline value after 120 minutes’ ischemia, but elevated to 100.4±29.4% higher when the reperfusion began. The ERNA remained 94.3±21.65% higher than baseline even after 60 minutes’ reperfusion. In the fluid expansion group, the ERNA also was 21.4±2.4% lower than the baseline, but still elevated to 29.3±5.2% higher even the hypotension were corrected by fluid expansion in the reperfusion injury. There was only mild change of afferent renal nerve activity (ARNA) in ischemia period. When the reperfusion period began, the ARNA was obviously depressed (37.5±5.8% lower than baseline) 。After 60 minutes’ reperfusion, the ARNA was still 45.7±8.1% lower than baseline. The fluid expansion did not change the depressed ARNA in reperfusion period. We also detected the levels of calcitonin-gene related pepetide (CGRP) in portal vein and intestinal tissue were higher than baseline values after 60 minutes’ reperfusion. (in portal vein: 92.2±4.4 pg/ml v.s 57.8±0.6 pg/ml, and in intestine: 655.8±115.9pg/mlv.s 60.5±9.4pg/ml), which implicated that CGRP was released into intestinal and portal vein from enteric nervous system in the reperfusion period. Fluid expansion not only lessens the hemodynamic changes and hemoconcentration occurring in reperfusion period, it also lessens the release of CGR, though the CGRP level was still higher than baseline value in reperfusion period. The level of another capsaicin-sensitive neuropeptide, substance P, was not found to be affected by this experiment model. Te result showed that ERNA increased after intestinal ischemia-reperfusion injury and the increased ERNA could reduce renal blood flow. To clarify the increased ERNA was due to baroreflex, another group of rats received fluid expansion before reperfusion began to correct the hemoconcentration and restore the renal blood flow. The amplitude of increased ERNA was lessen by fluid expansion, but was still higher than baseline, indicating the baroreflex could be totally responsible for the rise of ERNA in this model. Besides, a sensory impairment not related to baroreflex was recognized in intestinal ischemia-reperfusion injury for the depressed ARNA was not altered by fluid expansion. Through the inhibitory reno-renal reflex, the depressed ARNA could increased the contralateral ERNA. At the same time, the released CGRP in intestinal ischemia-reperfusion injury might directly stimulate the increase of ERNA. Our results indicated that intestinal ischemia-reperfusion injury causes a disturbance of renal nerve activity. The increased ERNA are related in part to hypotension and released CGRP in reperfusion period acting by a baroreflex way. Besides, the depressed ARNA leads to the loss of inhibitory action on contralateral ERNA, which further impairs the homeostasis of renal circulation and renal tubular function. To sum up, both the hypoxic and remote preconditioning effectively reduce the hepatic ischemia-reperfusion injury, partly due to the induction of heme oxygenase-1. The renal dysfunction in intestinal ischemia-reperfusion injury is in part due to an impairment of renal nerve activity induced in this injury model.

Includes bibliographical references (leaves 292-352)
xvi, 352 leaves
Determines the role of matrix metalloproteinases, MMP-2 and MMP-9 in reperfusion injury following skeletal muscle ischaemia; and, whether inhibition of MMPs by doxycycline protects against tissue damage.
Thesis (M.D.) -- University of Adelaide, Dept. of Surgery, 2002