Project description:Ischaemic preconditioning is a method of protecting tissue against ischaemia-reperfusion injury. It is an innate protective mechanism that increases a tissue's tolerance to prolonged ischaemia when it is first subjected to short burst of ischaemia and reperfusion. It is thought to provide this protection by increasing the tissue's tolerance to ischaemia, therby reducing oxidative stress, inflammation and apoptosis in the preconditioned tissue. We used microarrays to investigate the genomic response induced by ischaemic preconditioning in muscle biopsies taken from the operative leg of total knee arthroplasty patients in order to gain insight into the ischaemic preconditioning mechanism.
Project description:The transcriptional effects of urocortin I, urocortin II and tempol were compared to saline treatment in a rat model of in vivo coronary artery occlusion model of ischaemia/reperfusion injury of 25 min ischaemia and 2 hr reperfusion. <br>The treatment groups were as follows (i) sham operation or LAD occlusion with infusion of (ii) saline, (iii) 15 ?g/kg Ucn I, (iv) 15 ?g/kg Ucn II and (v) 100 mg/kg tempo infused just prior to reperfusionl.<br>Following 2 hr reperfusion the left ventricle was removed, snap frozen, followed by RNA extraction.
Project description:Ischaemic preconditioning is a method of protecting tissue against ischaemia-reperfusion injury. It is an innate protective mechanism that increases a tissue's tolerance to prolonged ischaemia when it is first subjected to short burst of ischaemia and reperfusion. It is thought to provide this protection by increasing the tissue's tolerance to ischaemia, therby reducing oxidative stress, inflammation and apoptosis in the preconditioned tissue. We used microarrays to investigate the genomic response induced by ischaemic preconditioning in muscle biopsies taken from the operative leg of total knee arthroplasty patients in order to gain insight into the ischaemic preconditioning mechanism. Patients undergoing primary knee arthroplasty were randomised to control and treatment (ischaemic preconditioning) groups. Patients in the treatment group received a preconditioning stimulus immediately prior to surgery. The ischaemic preconditioning stimulus consisted of three five-minute periods of tourniquet insufflation on the lower operative limb, interrupted by five minute periods of reperfusion. All patients had a tourniquet applied to the lower limb after the administration of spinal anaesthesia, as per normal protocol for knee arthroplasty surgery. Muscle biopsies were taken from the quadriceps muscle of the operative knee at the immediate onset of surgery (T0) and at 1 hour into surgery (T1). Total RNA was extracted from biospies of four control and four treatment patients and hybridised to the Affymetrix Human U133 2.0 chip.
Project description:In DCD organ donation one of the significant problems for the organ is the inflammatory response due to ischaemia reperfusion injury caused by warm ischaemia prior to retrieval followed by warm reoxygenation upon transplantation. We developed a model where a DCD kidney was retrieved and preserved using hypothermic machine perfusion with cold University of Wisconsin solution with/without the addition of heparinoids low molecular weight heparin and pentosan polysulfate (PPS) followed by warm oxygenated perfusion with modified Krebs-Henseleit buffer to simulate early ischaemia reperfusion injury (IRI). The donor rats consisted of a control group cold perfusion group compared to warm perfusion and groups treated with either low molecular weight heparin or PPS to assess their activity in ameliorating IRI. We used equimolar pooled RNA samples from each group to perform an exploratory microarray experiment Donor kidneys were harvested and preserved by hypothermic machine perfusion with University of Wisconsin (UW) Solution for 3 hours followed by warm oxygenated perfusion with modified Krebs-Henseleit buffer for 30 minutes to simulate transplantation and whole blood ischaemia reperfusion injury. The model was then used to test the anti-inflammatory properties of the glycosaminoglycan heparin and PPS when used as supplements into perfusate solutions. Half of the donor kidney was used to extract RNA using the Tri reagent and stored in RNA Later. The qualitative best three RNAs from each group were quantified by fluorimetry and mixed in an equimolar manner and used for microarray analysis.
Project description:In DCD organ donation one of the significant problems for the organ is the inflammatory response due to ischaemia reperfusion injury caused by warm ischaemia prior to retrieval followed by warm reoxygenation upon transplantation. We developed a model where a DCD kidney was retrieved and preserved using hypothermic machine perfusion with cold University of Wisconsin solution with/without the addition of heparinoids low molecular weight heparin and pentosan polysulfate (PPS) followed by warm oxygenated perfusion with modified Krebs-Henseleit buffer to simulate early ischaemia reperfusion injury (IRI). The donor rats consisted of a control group cold perfusion group compared to warm perfusion and groups treated with either low molecular weight heparin or PPS to assess their activity in ameliorating IRI. We used equimolar pooled RNA samples from each group to perform an exploratory microarray experiment
Project description:To reveal the alterations of mRNA profile in cerebral ischemia-reperfusion injury in rat. The SD rats were used to established the middle cerebral artery occlusion and reperfusion (MCAO/R) model. RNA-seq were performed to identify differences in gene expression.
Project description:In DCD organ donation one of the significant problems for the organ is the inflammatory response due to ischaemiua reperfusion injury caused by warm ischaemia prior to retrieval followed by warm reoxygenation upon transplantation. We developed a model where a DCD kidney was retrieved and stoired overnight then the organ was surgically attached to the femoral artery of a recipient rat and blood flow restored for up to six hours to simulate early ischaemia reperfusion injury (IRI). The recipient rats consisted of a control group compared to groups recieveing either low molecular weight heparin or an anti-inflammatory peptide to assess their activity in ameliorating IRI. We used equimolar pooled RNA samples from each group to perform an exploratory microarray experiment.
Project description:In DCD organ donation one of the significant problems for the organ is the inflammatory response due to ischaemiua reperfusion injury caused by warm ischaemia prior to retrieval followed by warm reoxygenation upon transplantation. We developed a model where a DCD kidney was retrieved and stoired overnight then the organ was surgically attached to the femoral artery of a recipient rat and blood flow restored for up to six hours to simulate early ischaemia reperfusion injury (IRI). The recipient rats consisted of a control group compared to groups recieveing either low molecular weight heparin or an anti-inflammatory peptide to assess their activity in ameliorating IRI. We used equimolar pooled RNA samples from each group to perform an exploratory microarray experiment. Donor kidneys were harvested and static cold stored in University of Wisconsin (UW) Solution overnight before being surgically connected to the femoral artery of a recipient rat to simulate transplantation and whole blood ischaemia reperfusion injury. The model was then used to test the anti-inflammatory properties of the glycosaminoglycan heparin and a peptide based upon the heparin binding domain of the pro-inflammatory chemokine CCL5. The treatments were delivered as a bolus in normal saline and control recipients were given normal saline as a control. The kidneys underwent perfusion by normal blood flow for a period of up to six hours after which the recipient was euthanased and the donor kidney was removed. Half of the donor kidney was used to extract RNA and half taken for immunohistochemical analysis. The RNA was extracted using the Tri reagent and stored in RNA Later. The qualitative best three RNAs from each group were quantified by fluorimetry and mixed in an equimolar manner and used for microarray analysis.
Project description:Ischemic stroke triggers severe focal hypoperfusion accompanied with deprivation of oxygen and glucose to the cerebral tissue, together with loss of ATP, depolorization of neurons, elevated extracellular potassium concentration, and subsequently leads to excitotoxicity as well as increased oxidative stress promoting microvascular injury, blood-brain-barrier deregulation, post-ischemic inflammation and eventually the consequential neurological deficit. Although reperfusion of ischemic brain tissue is critical for restoring normal function, it can paradoxically result in secondary damage, called ischemia/reperfusion (I/R) injury. Microarray analysis was performed on the right striatum and cortex (corresponded to infarct area) of post-I/R injured brain tissues of wild-type (WT-MCAO) using Illumina mouse Ref8 V2 genechips. Suture-induced middle cerebral artery occlusion was induced for 2h followed by reperfusion, with tissue extraction taking place 2h, 8h and 24h post-reperfusion (n=4 respectively). Sham controls were included in this study too (n=4 respectively).