Adenosine metabolized from extracellular ATP ameliorates organ injury by triggering A2BR signaling

BACKGROUND: Trauma and a subsequent hemorrhagic shock (T/HS) result in insufficient oxygen delivery to tissues and multiple organ failure. Extracellular adenosine, which is a product of the extracellular degradation of adenosine 5' triphosphate (ATP) by the membrane-embedded enzymes CD39 and CD73, is organ protective, as it participates in signaling pathways, which promote cell survival and suppress inflammation through adenosine receptors including the A2BR. The aim of this study was to evaluate the role of CD39 and CD73 delivering adenosine to A2BRs in regulating the host's response to T/HS. METHODS: T/HS shock was induced by blood withdrawal from the femoral artery in wild-type, global knockout (CD39, CD73, A2BR) and conditional knockout (intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl) mice. At 3 three hours after resuscitation, blood and tissue samples were collected to analyze organ injury. RESULTS: T/HS upregulated the expression of CD39, CD73, and the A2BR in organs. ATP and adenosine levels increased after T/HS in bronchoalveolar lavage fluid. CD39, CD73, and A2BR mimics/agonists alleviated lung and liver injury. Antagonists or the CD39, CD73, and A2BR knockout (KO) exacerbated lung injury, inflammatory cytokines, and chemokines as well as macrophage and neutrophil infiltration and accumulation in the lung. Agonists reduced the levels of the liver enzymes aspartate transferase and alanine transaminase in the blood, whereas antagonist administration or CD39, CD73, and A2BR KO enhanced enzyme levels. In addition, intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl mice showed increased intestinal injury compared to their wild-type VillinCre controls. CONCLUSION: In conclusion, the CD39-CD73-A2BR axis protects against T/HS-induced multiple organ failure

Effects of normobaric oxygen treatment on the brain injury and plasticity after newborn hypoxic ischemia

Doktora Tezi.Yenidoğan hipoksi iskemisi günümüzde klinik alanda halen en çok karşılaşılan durumlardan bir tanesidir. Bebeğin anne karnında oksijensiz kalması erken doğum olayı, doğum esnasında veya anne karnındayken kordon dolanması sonucu ortaya çıkmaktadır. Bu tür olaylar meydana geldiğinde bebeğin beynine yeteri kadar oksijen gidemez ve uzun dönemde epilepsi, mental bozukluklar gibi tedavisi zor sonuçların doğmasına sebep olabilir. Hipoksik iskeminin patofizyolojik süreçleri oksijensiz kalan dokuda hücre ölümlerine yol açmaktadır. Bu patofizyolojik süreçlerin engellenmesi kronik dönemde meydana gelebilecek telafisi mümkün olmayan sonuçların da önüne geçilmesine imkân tanıyacaktır. Normobarik oksijen tedavisi literatürde halen tartışılmakta olan bir konudur. İskemi sonrası dokuda oksijenlenmeyi arttırmakta, bununla beraber reaktif oksijen türlerinin de ortaya çıkmasına yol açmaktadır. Bu bağlamda yedi günlük sıçanlar anestezi altında sağ karotid arter bağlanmasını takiben, 2 saat süre ile %8 oksijene maruz bırakıldı. Hemen ardından sıçanlara farklı konsantrasyonlarda (%21- 70-100) oksijen tedavisi uygulandı. Yüksek oksijen konsantrasyonunun daha fazla serbest radikal üreteceği ve oksijen ile bir kümülatif etkisinin olup olmadığını anlayabilmek için ve iyi bir antioksidan olan melatonin %100 Oksijen grubu ile kombine edilerek ayrıca uygulandı. Daha sonra dekapite edilen hayvanların beyinleri hızla çıkartılıp 18 m kalınlığında kesitler alındı ve genel hasar dağılımı, yaşayan hücre sayısı, apopitotik hücre sayısı tayin edildi. Bunun yanında altta yatan patofizyolojinin daha iyi anlaşılabilmesi için hasarlı bölgeden alınan dokularda farklı protein konsantrasyonları ölçüldü. Ayrıca kronik dönemde hipoksi iskemi sonrası uygulanan tedavinin fonksiyonel geri kazanımlar üzerine etkisinin araştırılması için aydınlık-karanlık, rotarod, açık alan ve barnes labirenti testi uygulandı. Yapılan analizlere göre genel olarak artan oksijen konsantrasyonuna paralel olarak hücresel sağ kalımın arttığı, apopitotik hücre sayısının azaldığı, hücre ölüm mekanizmalarında görev alan proteinlerin inhibe olduğu görüldü. Ayrıca yapılan davranış testlerinin analizlerinde de yüksek oksijen konsantrasyonun özellikle melatonin ile kombine edilerek subakut dönemde anksiyeteyi azalttığı, motor koordinasyonu düzelttiği, hafıza ve öğrenme üzerine pozitif etkilerinin olduğu saptandı. Sonuç olarak hipoksi iskemi sonrası uygulanan normobarik %100 Oksijen-Melatonin kombinasyonunun insanlarda bir konsept çalışması olması planlanmaktadır.Newborn hypoxia is one of the most prevalent cases in the clinical status and can result from the hypoxia of the fetus in the womb. In such cases, blood supply is restricted to the fetus's brain, causing epilepsy or mental disorders that are difficult to treat in the long-term. Pathophsiological processes of hypoxic ischemia leads to cellular death in the hypoxic tissue. Inhibition of these pathophyisological processes will lead to the prevention of conditions in the chronical period. Normobaric oxygen treatment is still under debate in the literature. It has been reported that it increases oxygenation in the post-ischemic tissue along with the formation of reactive oxygen species. In this respect, seven days old pups were subjected to right carotid artery ligation, followed by 8% oxygen treatment for 2 hours. Then, pups were given different concentrations of oxygen treatment (21%, 70% or 100%). Hyperoxia was proposed to create increased free radicals and chosen to study whether there is a cumulative effect of this free radicals with oxygen. Moreoever, a strong antioxidant molecule, melatonin, was combined with 100% oxygen. The brains of the sacrificed animals were quickly removed and 18 um-thick sections were sliced. General damage area, surviving neurons and apoptotic cell numbers were analyzed in these sections. In addition, to understand the underlying pathophysiology, different protein concentrations were identified in the injured tissue. Moreoever, to investigate the effect of the treatment used following hypoxic ischemia on the functional recovery, lightdark, rotarod, open field and barnes maze tests were performed. In the light of the analyses carried out, we demonstrate that increasing oxygen concentration in general causes an increase in the cellular survival and a decrease in the number of apoptotic cells, while inhibiting the proteins involved in cellular death mechanisms. Moreover, it was shown to decrease anxiety, promote motor coordination and have positive effects on memory and learning during the subacute period when high oxygen concentration was combined with melatonin. In conclusion, normobaric 100% oxygen-melatonin combination following hypoxic ischemia could be a concept of proof study in humans

The investigation of plasticity related pathophysiological processes after cerebral ischemia using advance microscopy techniques

Beyin felci insanlarda en fazla motor fonksiyon bozukluklarına neden olan hastalıktır. Beyin felci sonrası post-iskemik fazda beynin endojen onarım mekanizmalarının etkinleştirilerek fonksiyonel olarak iyileşmesi, rehabilitasyon ve beyin plastisitesini uyaran tedaviler ile mümkün olabilmektedir. Post-iskemik fazda beyin felcinin iyileşmesi, kortikal ve subkortikal seviyede nöronal ağın yeniden organize edilmesiyle gerçekleşmektedir. Böylece fonksiyonel olmayan nöronal ağ, aktive edilip yeni bağlantılar ile güçlendirilerek hasarlı bölge çevresi ve beynin farklı bölgeleri ile iletişimi sağlanmaktadır. Beyin felci sonrasında hasarlı dokunun yeniden şekillendirilmesinde iskemik olmayan hemisferde (kontralateral) aksonal yapılanma (projeksiyon) gibi değişiklikler beynin, iskemik beyin felci sonrası oluşan yeni duruma hazırlanmasına ve fonksiyonel iyileşmeye katkıda bulunmaktadır. Daha önce bu konu ile ilgili yapılmış olan deneysel çalışmalar ipsilateral ve kontralateral hemisferlerde bu süreçlerin birbirinden bağımsız olarak geliştiğini göstermektedir. Ayrıca bu süreçlerin nasıl koordine edildiğine dair bilgiler yetersiz kalmaktadır. Bu tezin amacı kapsamında farelerde 30 dakikalık beyin felci uygulamasını takiben akut patofizyolojik değişikliklerin tamamlandıgı 3. günde sinaptik reoganizasyonların ve projeksiyonların üç boyutlu olarak belirlenmesi için hayvanların motor kortekslerine yeşil floresan ve kırmızı floresan ifade eden viral vektör enjeksiyonları yapıldı. Enjeksiyondan sonra 30. günde hayvanlar sakrifiye edilip beyinleri çıkartılarak 1 mm'lik bloklar halinde veya bütün beyin dokusu şeffaflaştırılıp, lazer taramalı konfokal mikroskop, multi foton mikroskop ve ışık perdeli mikroskop kullanılarak görüntülenerek hasar sonrası meydana gelen aksonal projeksiyonların 3 boyutlu haritalandırılması yapıldı. Sonuç olarak, şeffaflaştırma tekniğinin kullanılması ve bunun yanında viral vektör enjeksiyonlarının yapılması beyin felci patofizyolojisinin daha iyi anlaşılması, hasardan sonra meydana gelen plastisitenin kortiko spinal yolların daha iyi belirlenmesi ve son olarak geliştirilecek tedavi yöntemlerinin etkinliklerinin yorumlanmasına katkı sağlayacağı düşünülmektedir.Brain ischemia, is the leading cause of motor dysfunctions in humans. During the post-ischemic phase after brain ischemia, activation of endogenous repair mechanisms and functional recovery in the brain are achieved by the treatments promoting brain plasticity. Recovery from brain ischemia in the post-ischemic phase is achieved by the reorganization of neuronal networks in the cortical and subcortical levels. Thus, dysfunctional neuronal network is activated and enhanced by the new connections to promote the interaction between the peri-infarct zone and the different areas of the brain. In the process of remodeling of the injured tissue following brain ischemia, alterations such as axonal structuring in the non-ischemic hemisphere (contralateral) contributes to the adaptation of the brain to this new condition and to functional recovery. Previous experimental studies have shown that these processes develop independently from each other in the ipsilateral and contralateral hemispheres. Moreover, there is not enough information on how these processes are coordinated. In the scope of this thesis, to determine the 3D synaptic reorganization and projections, green and red fluorescent protein expressing viral vectors were injected at the 3rd day (when the acute pathophysiological alterations are completed) following 30 min brain ischemia in mice. Animals were sacrificed 30 days after the injection, brains were removed, 1 mm blocks of or whole brain tissue was cleared and the tissue was imaged using laser scanning confocal microscopy, multiphoton or lightsheet microscopy. Then, the axonal projections occurred after the injury was 3D mapped. In conclusion, it is believed that use of clearing technique as well as viral vector injections will help clarifying the pathophysiology of brain ischemia, identifying the corticospinal pathways of the plasticity after injury and finally interpreting the efficacy of future treatment methods

Advanced imaging techniques in neurological diseases

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Evidence that osteogenic and neurogenic differentiation capability of epidural adipose tissue-derived stem cells was more pronounced than in subcutaneous cells

Background/aim: The management of dura-related complications, such as the repairment of dural tears and reconstruction of large dural defects, remain the most challenging subjects of neurosurgery. Numerous surgical techniques and synthetic or autologous adjuvant materials have emerged as an adjunct to primary dural closure, which may result in further complications or side effects. Therefore, the subcutaneous autologous free adipose tissue graft has been recommended for the protection of the central nervous system and repairment of the meninges. In addition, human adipose tissue is also a source of multipotent stem cells. However, epidural adipose tissue seems more promising than subcutaneous because of the close location and intercellular communication with the spinal cord. Herein, it was aimed to define differentiation capability of both subcutaneous and epidural adipose tissue-derived stem cells (ASCs). Materials and methods: Human subcutaneous and epidural adipose tissue specimens were harvested from the primary incisional site and the lumbar epidural space during lumbar spinal surgery, and ASCs were isolated. Results: The results indicated that both types of ASCs expressed the cell surface markers, which are commonly expressed stem cells; however, epidural ASCs showed lower expression of CD90 than the subcutaneous ASCs. Moreover, it was demonstrated that the osteogenic and neurogenic differentiation capability of epidural adipose tissue-derived ASCs was more pronounced than that of the subcutaneous ASCs. Conclusion: Consequently, the impact of characterization of epidural ASCs will allow for a new understanding for dural as well as central nervous system healing and recovery after an injury.Marmara Universit

Evaluation of effects of UNC2025 on the traumatic brain injury

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Investigating the effects of memantine and melatonin treatment after traumatic brain injury in mice

Joint Congress of European Neurology -- MAY 31-JUN 03, 2014 -- Istanbul, TURKEYWOS: 000337563600554European Journal of Neurology…European Federation of Neurological Societie

A(2A) adenosine receptors regulate multiple organ failure after hemorrhagic shock in mice

Trauma hemorrhagic shock (T/HS) is a clinical condition that causes multiple organ failure that needs rapid intervention. Restricted oxygen at the cellular level causes inflammation and subsequent cell death. Adenosine triphosphate is the universal intracellular energy currency and an important extracellular inflammatory signaling molecule. Adenosine, an endogenous nucleotide formed as a result of the breakdown of adenosine triphosphate, is also released during T/HS. Adenosine binds to four G protein-coupled receptors (A1R, A2a, A2b, A3R) called adenosine receptors or P1 receptors. In the present study, we evaluated the effect of activation, inactivation, and genetic absence of A2aR (A2aR-/- mice) on T/HS-induced multiple organ failure. Wild-type mice were pretreated (30 min before shock induction) with an agonist or antagonist and then subjected to T/HS by withdrawing arterial blood and maintaining the blood pressure between 28 and 32 mm Hg. A2aR-/- mice were subjected to T/HS in the absence of pharmacologic treatment. Neutrophil sequestration was assessed by detecting myeloperoxidase, and Evans blue dye (EBD) method was used to analyze lung permeability. Blood and lung inflammatory cytokine levels were determined by sandwich enzyme-linked immunosorbent assay. The liver enzymes aspartate aminotransferase and alanine aminotransferase were determined spectrophotometrically from plasma. Activation of the apoptotic cascade was evaluated using a mouse apoptosis array. Our results demonstrate that the selective A2aR agonist CGS21680 decreases lung neutrophil sequestration, lung proinflammatory cytokines IL-6 and TNF-α, and bronchoalveolar lavage EBD. Pretreatment with the selective antagonist ZM241385 and genetic blockade in A2aR-/- mice increased neutrophil sequestration, proinflammatory cytokine levels, and bronchoalveolar lavage fluid EBD. The myeloperoxidase level in the lung was also increased in A2aR-/- mice. We observed that antiapoptotic markers decreased significantly with the absence of A2aR in the lung and spleen after T/HS. In conclusion, our data demonstrate that activation of A2aR regulates organ injury and apoptosis in the setting of T/HS.United States Department of Health & Human Services, National Institutes of Health (NIH) - US

Investigation the effects of memantin and melatonin treatment after traumatic brain injury in mice

Joint Congress of European Neurology -- MAY 31-JUN 03, 2014 -- Istanbul, TURKEYWOS: 000347674000553…European Federation of Neurological Association

Effects of normobaric oxygen and melatonin on reperfusion injury: Role of cerebral microcirculation

WOS: 000363185200028PubMed ID: 26416428In order to protect the brain before an irreversible injury occurs, penumbral oxygenation is the primary goal of current acute ischemic stroke treatment. However, hyperoxia treatment remains controversial due to the risk of free radical generation and vasoconstriction. Melatonin is a highly potent free radical scavenger that protects against ischemic stroke. Considering its anti-oxidant activity, we hypothesized that melatonin may augment the survival-promoting action of normobaric oxygen (NBO) and prevent brain infarction. Herein, we exposed mice to 30 or 90 min of intraluminal middle cerebral artery occlusion (MCAo) and evaluated the effects of NBO (70% or 100% over 90 min), administered either alone or in combination with melatonin (4 mg/ kg, i.p.), on disseminate neuronal injury, neurological deficits, infarct volume, blood-brain barrier (BBB) permeability, cerebral blood flow (CBF) and cell signaling. Both NBO and particularly melatonin alone reduced neuronal injury, neurological deficits, infarct volume and BBB permeability, and increased post-ischemic CBF, evaluated by laser speckle imaging (LSI). They also improved CBF significantly in the ischemic-core and penumbra, which was associated with reduced IgG extravasation, DNA fragmentation, infarct volume, brain swelling and neurological scores. Levels of phosphorylated Akt, anti-apoptotic Bcl-xL, pro-apoptotic Bax and endothelial nitric oxide synthase (NOS) were re-regulated after combined oxygen and melatonin delivery, whereas neuronal and inducible NOS, which were increased by oxygen treatment, were not influenced by melatonin. Our present data suggest that melatonin and NBO are promising approaches for the treatment of acute-ischemic stroke, which encourage proof-of-concept studies in human stroke patients.TUBITAK (The Scientific and Technological Research Council of Turkey) [111S418, 114S402]; Turkish Academy of Sciences (TUBA); EMBO (European Molecular Biology Organization) installation grantThis work was supported by TUBITAK (The Scientific and Technological Research Council of Turkey/111S418; 114S402), Turkish Academy of Sciences (TUBA) and EMBO (European Molecular Biology Organization) installation grant