Academic literature on the topic 'In vitro ischemia model'

Successful kidney transplantation is a life-saving procedure to patients with irreversible chronic renal failure. Despite the presence of various obstacles facing this surgery, preserving donor kidney and consequent ischemia reperfusion injury (IRI) are still major challenges affecting renal function as well as prognosis of transplant surgery. This study pursued two main aims: firstly, characterising changes in damage associated inflammatory gene expressions through developing, and analysis of an in vitro model of proximal tubular epithelial cells (PTEC) of normal human kidney mimicking renal IRI in vivo. The second aim was to simulate the concurrence of factors relevant to human intervention (renoprotective anaesthesia, peri- and postoperative analgesia, volume substitution) in mice deficient of properdin and congenic controls and to allow long-term observation of renal outcome after IRI. In this study, a reproducible and standardisable in vitro model was developed. It demonstrated the complexity of signalling where a multitude of factors affects the target cells. Secondly, the use of congenic properdin deficient mice showed that properdin has a significant role to play in renal injury (and recovery). There was significant impairment in renal function (and structure) compared to wildtype mice after IRI.

An interruption of the blood supply to the brain, as occurs during ischemic stroke, results in a rapid decline of ATP levels and a subsequent loss of neuronal function and viability. Under physiological conditions the brain reuses ATP degradation metabolites, such as hypoxanthine, via the purine salvage pathway, to restore its ATP pool. However, the massive degradation of ATP during ischemia results in the accumulation and loss of diffusible purine metabolites and thereby leads to a reduction in the post-ischemic ATP pool size, leaving the brain more vulnerable to secondary ischemic insults (recurrent strokes) and less able to deploy reparative mechanisms. The aim of this study was to improve the recovery of post-ischemic ATP levels by enhancing the purine salvage pathway, with substances that are already known to be tolerated in humans. Using acute hippocampal rat brain slices, I found that 1 mM Ribose (Rib) and 50 μM Adenine (Ade), two main metabolites of the purine salvage pathway, significantly increased the tissue ATP levels under basal conditions. Rib/Ade pre-treatment results in accelerated decline of synaptic transmission after onset of oxygen/glucose deprivation (OGD), due to increased adenosine release. However, this intervention does not delay the onset of anoxic depolarisation, or improve the recovery of synaptic transmission after prolonged ischemic periods. Pre-treatment of brain slices with 1 mM creatine, which increases phosphocreatine levels and thereby buffers the rapid decline of ATP levels upon energy shortage, significantly delays the onset of AD and helps to improve the recovery of synaptic transmission. By using cultured cerebellar granule cells, for more protracted studies on cell viability after OGD, I show that addition of Rib/Ade after ischemia helps to improves cell viability. Therefore my results suggest that both, delaying the decline of ATP upon onset of OGD (pre-treatment with creatine), or enhancing the post-ischemic recovery of ATP (post-treatment with Rib/Ade) are useful strategies to improve cell survival and function after in vitro ischemia.

Negli ultimi secoli, l'aspettativa di vita è aumentata grazie a uno stile di vita migliore, ma di conseguenza sono aumentate le patologie legate all'invecchiamento. L'invecchiamento è un processo fisiologico complesso e modificazioni legate all'età sono evidenti anatomicamente e fisiologicamente nella BEE. L'accumulo di danno ossidativo alle macromolecole da parte di RONS e ROS in BEE può essere cruciale nello sviluppo e nella progressione di diverse patologie del SNC. In questa situazione l'ischemia cerebrale potrebbe alterare ulteriormente l'equilibrio ossidante/antiossidante a favore degli ossidanti. In questo scenario, l'alimentazione può contrastare gli impatti ossidativi e le diete arricchite di polifenoli possono fornire effetti benefici. Il caffè è stato descritto come una fonte molto importante di composti antiossidanti (Ricci A. et al., 2018), ma la sua lavorazione produce ogni anno grosse quantità di scarto (Mussatto et al., 2011). Seguendo queste linee guida, lo scopo di questo studio era di valutare le proprietà antiossidanti di diversi composti correlati al caffè, da soli e combinati insieme, in un modello in vitro di ischemia. I composti utilizzati sono stati: fitoestratti derivanti dagli scarti della produzione del caffè e arricchiti in specifici componenti polifenolici; e metaboliti del caffè individuati nel plasma di persone che bevono caffè. Il momento successivo alla riossigenazione provoca un aumento dei ROS, raggiungendo un picco massimo entro 1h dal ripristino delle normali condizioni di coltura (Adibhatla RM et al., 2001). Pertanto, per la valutazione delle proprietà antiossidanti dopo OGD, come condizione di maggior stress è stato scelto l'intervallo 0-1h immediatamente successivo al recupero. Quindi, al fine di valutare le proprietà antiossidanti dei composti del caffè in OGD, sono state effettuate valutazioni sullo stato di fosforilazione delle chinasi Erk e Akt, che se attive promuovono la migrazione di Nrf2 nel nucleo, sui livelli della proteina Nrf2 e sulla sua distribuzione intracellulare, ed infine sui livelli proteici di HO-1, come una dei suoi geni bersaglio. Inoltre, è stata valutata anche la proteina Hsp70, che è coinvolta nel controllo del ripiegamento delle proteine ed infine, è stata misurata la produzione di malondialdeide (MDA) 24 ore dopo il recupero come marker di perossidazione lipidica. I risultati hanno dimostrato la capacità dei composti correlati al caffè di riuscire ad attivare la via di segnalazione Nrf2 in modo diverso e che solo i metaboliti modulavano positivamente Hsp70. I risultati dell'MDA hanno suggerito che la presenza dei composti antiossidanti, testati da soli o combinati, aveva avuto un effetto positivo sulla sua modulazione. I risultati, quindi, hanno dimostrato le proprietà antiossidanti dei fitoestratti e dei metaboliti specifici del caffè, suggerendo che le sostanze stimolano la risposta antiossidante attivando diverse vie, che combinate insieme potrebbero potenziare la difesa antiossidante. L'effetto antiossidante dei metaboliti potrebbe indicare che l'assunzione moderata di caffè giornaliera in soggetti anziani esposti all'invecchiamento e maggior rischio di insulto ischemico, potrebbe contribuire alla riduzione dello stress ossidativo limitando il danno da riperfusione in caso di attacchi ischemici. Queste difese potrebbero essere aumentate attraverso i fitoestratti derivati dal caffè ingeriti come integratori alimentari. Il riutilizzo di questa biomassa di scarto, avrebbe un impatto positivo sia sull'economia che sull'ecosistema, in quanto ridurrebbe notevolmente l'inquinamento.<br>Nowadays, the people get older and older thanks to a better life-style, but consequently, carrying on pathologies typical of the old age, included aging. The aging is a complex physiological process and age-related changes are evident anatomically and physiologically in the BBB. The accumulation of oxidative damage to macromolecules by RONS and ROS in BBB can be crucial in the development and progression of different CNS pathologies. In this situation, cerebral ischemia could further alter the oxidant/antioxidant balance in favour of oxidants. In this scenario, nutrition can counteract the oxidative impacts, polyphenol-enriched diets can provide beneficial effects, preventing cognitive decline and degenerative disorders. More recently, coffee has been described as a very important source of antioxidant compounds (Ricci A. et al., 2018) but its production generates large amount of waste. According to these guidelines, the aim of this study was to evaluate the antioxidant properties of several coffee-related compounds alone and combined together in an in vitro model of ischemia. The compounds used were: phytoextracts deriving from the waste of coffee production and enriched in specific polyphenolic components; and coffee metabolites found in plasma of people drinking coffee. The moment after reoxygenation causes a considerable increase in ROS, reaching a maximum peak within 1 hour of the restoration of normal culture conditions (Adibhatla RM et al., 2001). Therefore, for the evaluation of the antioxidant properties after OGD, the time span 0-1h immediately following recovery was chosen as the condition of greatest stress. Therefore, in order to evaluate the antioxidant properties of the coffee compound under OGD, the antioxidant pathway Nrf2 was analyzed within 0-1h, immediately following recovery. Evaluations were performed on the state of phosphorylation of Erk and Akt kinases, which if active promote Nrf2 migration in the nucleus, on the levels of the Nrf2 protein and on its intracellular distribution, and finally on the protein levels of HO-1, as one of its genes target. Furthermore, the protein Hsp70, which is involved in the control of protein folding, was also evaluated. Finally, malondialdehyde (MDA) production was measured as a marker of lipid peroxidation 24 hours after recovery. The results suggested the ability of coffee-related compounds to activate the Nrf2 signaling pathway differently and only the pre-treatment with metabolites modulated positively Hsp70. MDA results suggested that the presence of the antioxidant compounds, tested alone or combined, had a positive effect on its modulation. The results showed the antioxidant properties of phytoextracts and specific coffee metabolites, suggesting that the substances stimulate the antioxidant response by activating different pathways, which combined together in the mix, could enhance antioxidant defense. The antioxidant effect of coffee metabolites could indicate that the moderate intake of coffee every day in elderly subjects exposed to aging and greater risk of ischemic insult, could contribute to the reduction of oxidative stress by limiting reperfusion damage in the case of ischemic attacks. These defenses could be increased through the phytoextracts derived from coffee ingested as food supplements. The reuse of this waste biomass, would have a positive impact on both the economy and the ecosystem, as it would significantly reduce pollution.

L’ictus o accident cerebrovascular és la segona causa de mort en els països industrialitzats i constitueix la primera causa de discapacitat en adults. L’únic tractament aprovat en l’actualitat és el trombolític activador del plasminògen tissular (tPA), el qual només es pot aplicar en un nombre molt reduït de pacients i dintre d’una estreta finestra terapèutica. Els mecanismes de mort cel·lular en la isquèmia cerebral són amplis i venen provocats per l’interrupció del flux sanguini al cervell, el qual provoca una mort ràpida i eminentment necròtica en el nucli de la zona afectada i una mort de caràcter apoptòtic i més lenta al voltant, en la zona de penombra. El gran impacte socio-econòmic de la malaltia i l’existència d’una mort programada dil·latada en el temps han fet que es posin grans esforços en la búsqueda de mecanismes per salvar la zona de penombra. Tenint en compte aquests fets, el present treball s’ha centrat en l’estudi dels mecanismes de mort cel·lular involucrats en la isquèmia cerebral. Per fer-ho, s’ha utilitzat un model de privació d’oxigen i glucosa (OGD) en cultius corticals mixtes d’embrions de rata. Utilitzant aquest model d’isquèmia s’ha observat neuroprotecció pel bloqueig dels receptors NMDA, principal responsable de l’entrada massiva de calci, i l’activació de la caspasa-3, una proteasa encarregada del desmantellament cel·lular durant l’apoptosis. A més, s’ha estudiat el paper dels antagonistes dels receptors de mort en l’OGD. Aquests receptors són responsables de l’activació de la via apoptòtica extrínseca. S’ha observat que l’OGD indueix la degradació dels antagonistes FLIPL i IAP2 i modula l’expressió de FAIML a través de la via de les MAP cinases. Per altre banda, s’ha observat que el silenciament o la sobrexpressió de FAIML mitjançant vectors lentivirals no afecta la viabilitat dels cultius així com tampoc la morfologia nuclear apoptòtica ni els nivells de caspasa-3 activa en les neurones sotmeses a l’insult isquèmic. En conjunt aquests resultats han servit per aprofundir en els mecanismes moleculars implicats en la isquèmia cerebral i poden servir de base per futurs estudis que ajudin al disseny de noves estrategies terapèutiques.<br>Stroke is the second cause of death in industrialized countries and is the leading cause of disability in adults. The only currently approved treatment is the thrombolytic tissue plasminogen activator (tPA), which can be applied only in a very small number of patients and within a narrow therapeutic window. The mechanisms of cell death in brain ischemia are numerous and are caused by the interruption of the blood flow to the brain, which causes a quick necrotic death in the core of the affected area and a slow apoptotic-like death around, in the ischemic penumbra. The major socio-economic impact of the disease and the existence of a programmed cell death that stretches through time explain the effort that is being done to find new strategies to save the penumbra. Given these facts, the present work has focused on studying the mechanisms of cell death involved in brain ischemia. To do this, we used a model of oxygen and glucose deprivation (OGD) in mixed cortical cultures from rat embryos. Using this model of ischemia we observed neuroprotection by blocking NMDA receptor, the primarily responsible for the massive influx of calcium during ischemia, and activation of caspase-3, a protease responsible for dismantling the cell during apoptosis. In addition, we studied the role of death receptor antagonists in OGD. These receptors are responsible for the activation of the extrinsic apoptotic pathway. It has been observed that OGD induces degradation of the antagonists FLIPL and IAP2 and modulate the expression of FAIML through the MAP kinase pathway. On the other hand, we observed that the overexpression or silencing of FAIML using lentiviral vectors did not affect the viability of the cultures nor the apoptotic nuclear morphology or the levels of active caspase-3 in the neurons subjected to the ischemic insult. Together these results have served to study the molecular mechanisms involved in brain ischemia and may provide the basis for future studies that will help to design new therapeutic strategies.

Title: Role and potential of endothelial progenitor cells in cerebral vasospasm Abstract: Background and aim: Despite many treatment approaches, cerebral vasospasm and delayed ischemic neuronal damage (DIND) still represent a serious threat to patients with subarachnoid haemorrhage (SAH). Endothelial progenitor cells (EPC) have been involved as prognostic indicators in several vascular diseases and mesenchymal stem cells already have shown some benefits in ischemic injury. Aim of this study was to investigate the therapeutic potential of endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) in attenuating or preventing vasospasm and DIND in patients with SAH. Methods: Given the emergent role of DIND as a result of multifactorial hypoperfusion stress in the outcome of SAH patients, the efficacy of EPC and MSC in reducing neuronal damage has been evaluated in an in vitro model of ischemia, namely the oxygen glucose deprivation (OGD), on primary rat cortical neuronal cultures. Further, we tested in a clinical observational study SAH patients with and without vasospasm for different recruitment patterns of circulating EPC. To this purpose arterial blood samples were collected at various timepoints from admission to discharge of the patients. On these samples real-time quantitative PCR (RT-qPCR) was performed to detect gene expression relative to EPCs, since cytofluorimetric analysis appeared not sensitive enough to detect this rare cell population. Results: Though present results need further confirmation, in vitro it was observed that both MSC and EPC treatment through conditioned medium or co-culture in transwell- although with some differences - mediate a survival advantage for OGD stressed neurons. Furthermore stem cell mediated treatment showed efficacy even when applied 24 hours after OGD stress induction. RT-qPCR results from a small sample of SAH patients might indicate an early mobilization of EPC related gene expression in subjects that do not develop vasospasm with a peak around day 4, whereas the expression of these genes remain invariably low in patients that develop vasospasm as in controls not affected by SAH. Conclusions: MSCs and EPCs seem to have an important potential role in preventing DIND in vitro as well as EPC recruitment might associate with lack of vasospasm in SAH patients. Further studies are needed to confirm these results and to prove a causal relationship between EPCs and vasospasm protection.