Publications by authors named "Muge Yemisci"

38 Publications

Updated review on the link between cortical spreading depression and headache disorders.

Expert Rev Neurother 2021 Jun 23. Epub 2021 Jun 23.

Department of Neurology and Algology, Gazi University Faculty of Medicine, Besevler, Ankara, Turkey.

Introduction: Experimental animal studies have revealed mechanisms that link cortical spreading depression (CSD) to the trigeminal activation mediating lateralized headache. However, conventional CSD as seen in lissencephalic brain is insufficient to explain some clinical features of aura and migraine headache.

Areas Covered: The importance of CSD in headache development including dysfunction of the thalamocortical network, neuroinflammation, calcitonin gene-related peptide, transgenic models, and the role of CSD in migraine triggers, treatment options, neuromodulation and future directions are reviewed.

Expert Opinion: The conventional understanding of CSD marching across the hemisphere is invalid in gyrencephalic brains. Thalamocortical dysfunction and interruption of functional cortical network systems by CSD, may provide alternative explanations for clinical manifestations of migraine phases including aura. Not all drugs showing CSD blocking properties in lissencephalic brains, have efficacy in migraine headache and monoclonal antibodies against CGRP ligand/receptors which are effective in migraine treatment, have no impact on aura in humans or CSD properties in rodents. Functional networks and molecular mechanisms mediating and amplifying the effects of limited CSD in migraine brain remain to be investigated to define new targets.
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http://dx.doi.org/10.1080/14737175.2021.1947797DOI Listing
June 2021

Migraine and neuroinflammation: the inflammasome perspective.

J Headache Pain 2021 Jun 10;22(1):55. Epub 2021 Jun 10.

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Background: Neuroinflammation has an important role in the pathophysiology of migraine, which is a complex neuro-glio-vascular disorder. The main aim of this review is to highlight findings of cortical spreading depolarization (CSD)-induced neuroinflammatory signaling in brain parenchyma from the inflammasome perspective. In addition, we discuss the limited data of the contribution of inflammasomes to other aspects of migraine pathophysiology, foremost the activation of the trigeminovascular system and thereby the generation of migraine pain.

Main Body: Inflammasomes are signaling multiprotein complexes and key components of the innate immune system. Their activation causes the production of inflammatory cytokines that can stimulate trigeminal neurons and are thus relevant to the generation of migraine pain. The contribution of inflammasome activation to pain signaling has attracted considerable attention in recent years. Nucleotide-binding domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) is the best characterized inflammasome and there is emerging evidence of its role in a variety of inflammatory pain conditions, including migraine. In this review, we discuss, from an inflammasome point of view, cortical spreading depolarization (CSD)-induced neuroinflammatory signaling in brain parenchyma, the connection with genetic factors that make the brain vulnerable to CSD, and the relation of the inflammasome with diseases that are co-morbid with migraine, including stroke, epilepsy, and the possible links with COVID-19 infection.

Conclusion: Neuroinflammatory pathways, specifically those involving inflammasome proteins, seem promising candidates as treatment targets, and perhaps even biomarkers, in migraine.
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http://dx.doi.org/10.1186/s10194-021-01271-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192049PMC
June 2021

Pericyte morphology and function.

Histol Histopathol 2021 Jun 17;36(6):633-643. Epub 2021 Feb 17.

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

The proper delivery of blood is essential for healthy neuronal function. The anatomical substrate for this precise mechanism is the neurovascular unit, which is formed by neurons, glial cells, endothelia, smooth muscle cells, and pericytes. Based on their particular location on the vessel wall, morphology, and protein expression, pericytes have been proposed as cells capable of regulating capillary blood flow. Pericytes are located around the microvessels, wrapping them with their processes. Their morphology and protein expression substantially vary along the vascular tree. Their contractibility is mediated by a unique cytoskeleton organization formed by filaments of actin that allows pericyte deformability with the consequent mechanical force transferred to the extracellular matrix for changing the diameter. Pericyte ultrastructure is characterized by large mitochondria likely to provide energy to regulate intracellular calcium concentration and fuel contraction. Accordingly, pericytes with compromised energy show a sustained intracellular calcium increase that leads to persistent microvascular constriction. Pericyte morphology is highly plastic and adapted for varying contractile capability along the microvascular tree, making pericytes ideal cells to regulate the capillary blood flow in response to local neuronal activity. Besides the vascular regulation, pericytes also play a role in the maintenance of the blood-brain/retina barrier, neovascularization and angiogenesis, and leukocyte transmigration. Here, we review the morphological and functional features of the pericytes as well as potential specific markers for the study of pericytes in the brain and retina.
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http://dx.doi.org/10.14670/HH-18-314DOI Listing
June 2021

F-actin polymerization contributes to pericyte contractility in retinal capillaries.

Exp Neurol 2020 10 29;332:113392. Epub 2020 Jun 29.

Hacettepe University, Institute of Neurological Sciences and Psychiatry, Ankara, Turkey. Electronic address:

Although it has been documented that central nervous system pericytes are able to contract in response to physiological, pharmacological or pathological stimuli, the underlying mechanism of pericyte contractility is incompletely understood especially in downstream pericytes that express low amounts of alpha-smooth muscle actin (α-SMA). To study whether pericyte contraction involves F-actin polymerization as in vascular smooth muscle cells, we increased retinal microvascular pericyte tonus by intravitreal injection of a vasoconstrictive agent, noradrenaline (NA). The contralateral eye of each mouse was used for vehicle injection. The retinas were rapidly extracted and fixed within 2 min after injections. Polymeric/filamentous (F-actin) and monomeric/globular (G-actin) forms of actin were labeled by fluorescently-conjugated phalloidin and deoxyribonuclease-I, respectively. We studied 108 and 83 pericytes from 6 NA- and 6 vehicle-treated retinas and, found that F/G-actin ratio, a microscopy-based index of F-actin polymerization, significantly increased in NA-treated retinas [median (IQR): 4.2 (3.1) vs. 3.5 (2.1), p = .006], suggesting a role for F-actin polymerization in pericyte contractility. Shift from G-actin monomers to polymerized F-actin was more pronounced in 5th and 6th order contracted pericytes compared to non-contracted ones [7.6 (4.7) vs. 3.2 (1.2), p < .001], possibly due to their dependence on de novo F-actin polymerization for contractile force generation because they express α-SMA in low quantities. Capillaries showing F-actin polymerization had significantly reduced diameters compared to the ones that did not exhibit increased F/G-actin ratio in pericytes [near soma / branch origin diameter; 0.67 (0.14) vs. 0.81 (0.34), p = .005]. NA-responsive capillaries generally did not show nodal constrictions but a tide-like diameter decrease, reaching a maximum near pericyte soma. These findings suggest that pericytes on high order downstream capillaries have F-actin-mediated contractile capability, which may contribute to the vascular resistance and blood flow regulation in capillary bed.
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http://dx.doi.org/10.1016/j.expneurol.2020.113392DOI Listing
October 2020

Post-stroke inflammatory response is linked to volume loss in the contralateral hemisphere.

J Neuroimmunol 2020 07 21;344:577247. Epub 2020 Apr 21.

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey. Electronic address:

Objectives: There is a delicate homeostatic balance between the central nervous system and immune system. Stroke triggers an immunodepressive state to suppress a potential immune reaction directed against neuroglial tissue; however, this supposedly protective response inadvertently results in an infection-prone, and thereby a pro-inflammatory setting. In this study, we assessed the magnitude of cerebral volume loss in the unaffected contralateral hemisphere following stroke, and determined its relationship with inflammatory cascades.

Methods: The volume of the hemisphere contralateral to the ischemic insult was measured on admission and follow-up MRI's in 50 ischemic stroke patients. Information related to clinical features, infectious complications, and markers of inflammation (erythrocyte sedimentation rate, neutrophil/lymphocyte ratio, C-reactive protein) were prospectively collected, and their relationship with hemispheric volume change was evaluated using bivariate and multivariate statistics.

Results: The contralateral hemisphere volume decreased by a median (interquartile range) of 14 (4-32) mL after a follow-up duration of 101 (63-123) days (p < .001); the volume reduction was 0.8 (0.2-1.8) % per month with respect to baseline. Old age, atrial fibrillation, stroke severity, C-reactive protein level, neutrophil/lymphocyte ratio, and development of infections during hospitalization were significantly associated with volume loss (p < .05). Stroke severity (NIHSS score or infarct volume) and inflammation related parameters (neutrophil/lymphocyte ratio or systemic infections) remained independently and positively associated with volume loss in multivariate regression models.

Conclusions: Cerebral tissue changes following stroke are not limited to the ischemic hemisphere. Apart from stroke severity, a pro-inflammatory state and post-stroke infections contribute to cerebral volume loss in the non-ischemic hemisphere.
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http://dx.doi.org/10.1016/j.jneuroim.2020.577247DOI Listing
July 2020

Retinal ischemia induces α-SMA-mediated capillary pericyte contraction coincident with perivascular glycogen depletion.

Acta Neuropathol Commun 2019 08 20;7(1):134. Epub 2019 Aug 20.

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Sihhiye, 06100, Ankara, Turkey.

Increasing evidence indicates that pericytes are vulnerable cells, playing pathophysiological roles in various neurodegenerative processes. Microvascular pericytes contract during cerebral and coronary ischemia and do not relax after re-opening of the occluded artery, causing incomplete reperfusion. However, the cellular mechanisms underlying ischemia-induced pericyte contraction, its delayed emergence, and whether it is pharmacologically reversible are unclear. Here, we investigate i) whether ischemia-induced pericyte contractions are mediated by alpha-smooth muscle actin (α-SMA), ii) the sources of calcium rise in ischemic pericytes, and iii) if peri-microvascular glycogen can support pericyte metabolism during ischemia. Thus, we examined pericyte contractility in response to retinal ischemia both in vivo, using adaptive optics scanning light ophthalmoscopy and, ex vivo, using an unbiased stereological approach. We found that microvascular constrictions were associated with increased calcium in pericytes as detected by a genetically encoded calcium indicator (NG2-GCaMP6) or a fluoroprobe (Fluo-4). Knocking down α-SMA expression with RNA interference or fixing F-actin with phalloidin or calcium antagonist amlodipine prevented constrictions, suggesting that constrictions resulted from calcium- and α-SMA-mediated pericyte contractions. Carbenoxolone or a Cx43-selective peptide blocker also reduced calcium rise, consistent with involvement of gap junction-mediated mechanisms in addition to voltage-gated calcium channels. Pericyte calcium increase and capillary constrictions became significant after 1 h of ischemia and were coincident with depletion of peri-microvascular glycogen, suggesting that glucose derived from glycogen granules could support pericyte metabolism and delay ischemia-induced microvascular dysfunction. Indeed, capillary constrictions emerged earlier when glycogen breakdown was pharmacologically inhibited. Constrictions persisted despite recanalization but were reversible with pericyte-relaxant adenosine administered during recanalization. Our study demonstrates that retinal ischemia, a common cause of blindness, induces α-SMA- and calcium-mediated persistent pericyte contraction, which can be delayed by glucose driven from peri-microvascular glycogen. These findings clarify the contractile nature of capillary pericytes and identify a novel metabolic collaboration between peri-microvascular end-feet and pericytes.
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http://dx.doi.org/10.1186/s40478-019-0761-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701129PMC
August 2019

Pericytes in Ischemic Stroke.

Adv Exp Med Biol 2019 ;1147:189-213

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.

Recent stroke research has shifted the focus to the microvasculature from neuron-centric views. It is increasingly recognized that a successful neuroprotection is not feasible without microvascular protection. On the other hand, recent studies on pericytes, long-neglected cells on microvessels have provided insight into the regulation of microcirculation. Pericytes play an essential role in matching the metabolic demand of nervous tissue with the blood flow in addition to regulating the development and maintenance of the blood-brain barrier (BBB), leukocyte trafficking across the BBB and angiogenesis. Pericytes appears to be highly vulnerable to injury. Ischemic injury to pericytes on cerebral microvasculature unfavorably impacts the stroke-induced tissue damage and brain edema by disrupting microvascular blood flow and BBB integrity. Strongly supporting this, clinical imaging studies show that tissue reperfusion is not always obtained after recanalization. Therefore, prevention of pericyte dysfunction may improve the outcome of recanalization therapies by promoting microcirculatory reperfusion and preventing hemorrhage and edema. In the peri-infarct tissue, pericytes are detached from microvessels and promote angiogenesis and neurogenesis, and hence positively effect stroke outcome. Expectedly, we will learn more about the place of pericytes in CNS pathologies including stroke and devise approaches to treat them in the next decades.
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http://dx.doi.org/10.1007/978-3-030-16908-4_9DOI Listing
August 2019

Aura and Stroke: relationship and what we have learnt from preclinical models.

J Headache Pain 2019 May 29;20(1):63. Epub 2019 May 29.

Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Background: Population-based studies have highlighted a close relationship between migraine and stroke. Migraine, especially with aura, is a risk factor for both ischemic and hemorrhagic stroke. Interestingly, stroke risk is highest for migraineurs who are young and otherwise healthy.

Main Body: Preclinical models have provided us with possible mechanisms to explain the increased vulnerability of migraineurs' brains towards ischemia and suggest a key role for enhanced cerebral excitability and increased incidence of microembolic events. Spreading depolarization (SD), a slowly propagating wave of neuronal depolarization, is the electrophysiologic event underlying migraine aura and a known headache trigger. Increased SD susceptibility has been demonstrated in migraine animal models, including transgenic mice carrying human mutations for the migraine-associated syndrome CADASIL and familial hemiplegic migraine (type 1 and 2). Upon experimentally induced SD, these mice develop aura-like neurological symptoms, akin to patients with the respective mutations. Migraine mutant mice also exhibit an increased frequency of ischemia-triggered SDs upon experimental stroke, associated with accelerated infarct growth and worse outcomes. The severe stroke phenotype can be explained by SD-related downstream events that exacerbate the metabolic mismatch, including pericyte contraction and neuroglial inflammation. Pharmacological suppression of the genetically enhanced SD susceptibility normalizes the stroke phenotype in familial hemiplegic migraine mutant mice. Recent epidemiologic and imaging studies suggest that these preclinical findings can be extrapolated to migraine patients. Migraine patients are at risk for particularly cardioembolic stroke. At the same time, studies suggest an increased incidence of coagulopathy, atrial fibrillation and patent foramen ovale among migraineurs, providing a possible path for microembolic induction of SD and, in rare instances, stroke in hyperexcitable brains. Indeed, recent imaging studies document an accelerated infarct progression with only little potentially salvageable brain tissue in acute stroke patients with a migraine history, suggesting an increased vulnerability towards cerebral ischemia.

Conclusion: Preclinical models suggest a key role for enhanced SD susceptibility and microembolization to explain both the occurrence of migraine attacks and the increased stroke risk in migraineurs. Therapeutic targeting of SD and microembolic events, or potential causes thereof, will be promising for treatment of aura and may also prevent ischemic infarction in vulnerable brains.
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http://dx.doi.org/10.1186/s10194-019-1016-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6734247PMC
May 2019

Brain Peptides for the Treatment of Neuropsychiatric Disorders.

Curr Pharm Des 2018 ;24(33):3905-3917

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

The realization of the importance of growth factors in adult CNS led to several studies investigating their roles in neuropsychiatric disorders. Based on the observations that chronic stress decreases brain-derived neurotrophic factor (BDNF) and antidepressant treatments reverse BDNF to normal levels, "neurotrophic hypothesis of depression" was proposed. Subsequent studies found that several other growth factors, including fibroblast growth factor (FGF), vascular endothelial growth factor, nerve growth factor were also decreased by chronic stress. Growth factors promote stem cell survival, angiogenesis and neurogenesis in addition to having anti-apoptotic and anti-inflammatory effects, all of which make them potential drug candidates as neuroprotective or neurorestorative agents. Indeed, certain peptides have consistently been shown to improve stroke outcome in experimental models of cerebral ischemia. Recent developments in nanotechnology appear promising in overcoming the blood-brain barrier and in delivering sufficient amounts of these large peptides to the brain after systemic administration. In addition to the translational potential resulting from application of nanotechnical approaches for delivering these large peptide growth factors, recent success obtained with small molecule and peptide antagonists of calcitonin gene-related peptide has created renewed enthusiasm to elucidate the role of neuropeptides in migraine headache, one of the most common health problems in the world. In this review, we will first focus on the role of FGF2 in mood disorders as well as in ischemic stroke. We will also introduce the nanomedicines developed to efficiently deliver FGF2 to the brain. In the last section, we will explore roles of the neuropeptides in migraine and its acute and prophylactic treatment.
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http://dx.doi.org/10.2174/1381612824666181112112309DOI Listing
November 2019

Nuclear expansion and pore opening are instant signs of neuronal hypoxia and can identify poorly fixed brains.

Sci Rep 2018 10 3;8(1):14770. Epub 2018 Oct 3.

Hacettepe University, Institute of Neurological Sciences and Psychiatry, Ankara, 06100, Turkey.

The initial phase of neuronal death is not well characterized. Here, we show that expansion of the nuclear membrane without losing its integrity along with peripheralization of chromatin are immediate signs of neuronal injury. Importantly, these changes can be identified with commonly used nuclear stains and used as markers of poor perfusion-fixation. Although frozen sections are widely used, no markers are available to ensure that the observed changes were not confounded by perfusion-induced hypoxia/ischemia. Moreover, HMGB1 was immediately released and p53 translocated to mitochondria in hypoxic/ischemic neurons, whereas nuclear pore complex inhibitors prevented the nuclear changes, identifying novel neuroprotection targets.
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http://dx.doi.org/10.1038/s41598-018-32878-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170374PMC
October 2018

Data of ascending cortical vein occlusion induced spreading depression.

Data Brief 2018 Jun 18;18:1462-1465. Epub 2018 Apr 18.

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

The data presented in this article are related to the research article entitled "Microembolism of single cortical arterioles can induce spreading depression and ischemic injury; a potential trigger for migraine and related MRI lesions" (Donmez-Demir et al., 2018) [1]. This article presents data showing that thrombosis of a small ascending cortical vein (25 µm) in the mouse may also trigger spreading depression as does penetrating arteriole occlusion, although less frequently (22% vs. 100%).
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http://dx.doi.org/10.1016/j.dib.2018.04.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997957PMC
June 2018

Improving Microcirculatory Reperfusion Reduces Parenchymal Oxygen Radical Formation and Provides Neuroprotection.

Stroke 2018 05 18;49(5):1267-1275. Epub 2018 Apr 18.

From the Institute of Neurological Sciences and Psychiatry (A.T.-S., M.Y., Y.G.-O., S.E.E., H.K., T.D.)

Background And Purpose: Reperfusion is the most significant determinant of good outcome after ischemic stroke. However, complete reperfusion often cannot be achieved, despite satisfactory recanalization. We hypothesized that microvascular protection was essential for achieving effective reperfusion and, hence, neuroprotection. To test this hypothesis, we have developed an in vivo model to differentially monitor parenchymal and vascular reactive oxygen species (ROS) formation. By comparing the ROS-suppressing effect of N-tert-butyl-α-phenylnitrone (PBN) with its blood-brain barrier impermeable analog 2-sulfo-phenyl-N-tert-butylnitrone (S-PBN), we assessed the impact of vascular ROS suppression alone on reperfusion and stroke outcome after recanalization.

Methods: The distal middle cerebral artery was occluded for 1 hour by compressing with a micropipette and then recanalized (n=60 Swiss mice). ROS formation was monitored for 1 hour after recanalization by intravital fluorescence microscopy in pial vasculature and cortical parenchyma with topically applied hydroethidine through a cranial window. PBN (100 mg/kg) or S-PBN (156 mg/kg) was administered shortly before recanalization, and suppression of the vascular and parenchymal hydroethidine fluorescence was examined (n=22). Microcirculatory patency, reperfusion, ischemic tissue size, and neurological outcome were also assessed in a separate group of mice 1 to 72 hours after recanalization (n=30).

Results: PBN and S-PBN completely suppressed the reperfusion-induced increase in ROS signal within vasculature. PBN readily suppressed ROS produced in parenchyma by 88%. S-PBN also suppressed the parenchymal ROS by 64% but starting 40 minutes later. Intriguingly, PBN and S-PBN comparably reduced the size of ischemic area by 65% and 48% (>0.05), respectively. S-PBN restored the microvascular patency and perfusion after recanalization, suggesting that its delayed parenchymal antioxidant effect could be secondary to improved microcirculatory reperfusion.

Conclusions: Promoting microvascular reperfusion by protecting vasculature can secondarily reduce parenchymal ROS formation and provide neuroprotection. The model presented can be used to directly assess pharmacological end points postulated in brain parenchyma and vasculature in vivo.
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http://dx.doi.org/10.1161/STROKEAHA.118.020711DOI Listing
May 2018

Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection.

Elife 2018 03 21;7. Epub 2018 Mar 21.

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling.
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http://dx.doi.org/10.7554/eLife.34861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862523PMC
March 2018

Metabolomic Estimation of the Diagnosis and Onset Time of Permanent and Transient Cerebral Ischemia.

Mol Neurobiol 2018 Jul 21;55(7):6193-6200. Epub 2017 Dec 21.

Clinical Neurosciences Group, Institut de Recerca Biomèdica de Lleida (IRBLleida), Universitat de Lleida, Lleida, Spain.

Determining the time of stroke onset in order to apply recanalization therapies within the accepted therapeutic window and the correct diagnosis of transient ischemic attack (TIA) are two common clinical problems in acute cerebral ischemia management. Therefore, biomarkers helping in this conundrum could be very helpful. We developed mouse models of distal middle cerebral artery occlusion mimicking TIA and ischemic stroke (IS), respectively. Plasma samples were analyzed by metabolomics at 6, 12, 24, and 48 h post onset in order to find TIA- and time-related stroke biomarkers. The results were validated in a second experimental cohort. Plasma metabolomic profiles identified time after stroke events with a very high accuracy. Specific metabolites pointing to a recent event (< 6 h) were identified. A multivariate (partial least square discriminant analyses [PLS-DA]) model was also able to separate samples from TIA, IS, and sham events with high accuracy and to obtain specific metabolites for each time point. The combination of mice models of focal ischemia with plasma metabolomics allows the discovery of candidate biomarkers for the diagnosis and estimation of onset time of stroke and TIA diagnosis.
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http://dx.doi.org/10.1007/s12035-017-0827-5DOI Listing
July 2018

Preparation and Characterization of Biocompatible Chitosan Nanoparticles for Targeted Brain Delivery of Peptides.

Methods Mol Biol 2018 ;1727:443-454

Faculty of Medicine, Department of Neurology, Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Here, we describe a nanocarrier system that can transfer chitosan nanoparticles loaded with either small peptides such as the caspase inhibitor Z-DEVD-FMK or a large peptide like basic fibroblast growth factor across the blood-brain barrier. The nanoparticles are selectively directed to the brain and are not measurably taken up by the liver and spleen. Intravital fluorescent microscopy provides an opportunity to study the penetration kinetics of nanoparticles loaded with fluorescent agents such as Nile red. Nanoparticles functionalized with anti-transferrin antibody and loaded with peptides efficiently provided neuroprotection when systemically administered either as a formulation bearing a single peptide or a mixture of them. Failure of brain permeation of the nanoparticles after inhibition of vesicular transcytosis by imatinib as well as when nanoparticles were not functionalized with anti-transferrin antibody indicates that this nanomedicine formulation is rapidly transported across the blood-brain barrier by receptor-mediated transcytosis.
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http://dx.doi.org/10.1007/978-1-4939-7571-6_36DOI Listing
July 2018

Microembolism of single cortical arterioles can induce spreading depression and ischemic injury; a potential trigger for migraine and related MRI lesions.

Brain Res 2018 01 26;1679:84-90. Epub 2017 Nov 26.

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey; Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey; Neuroscience Center, Massachusetts General Hospital, Harvard University, Boston, USA. Electronic address:

Increasing epidemiological evidence suggests an association between migraine with aura (MA) and cardiovascular events. There is experimental as well as clinical evidence implying cerebral microembolism as a potential trigger for MA attacks. Microembolism may also account for some of the ischemic MRI lesions more commonly observed in MA than in general population. Limited size of clinically-silent MRI lesions suggests isolated occlusion of a small vessel. However, it is not known whether selective thrombosis of a small arteriole (e.g. single mouse penetrating arteriole - PA), can induce cortical spreading depression (CSD), the putative cause of migraine aura and, hence, trigger an MA attack. For this, we mimiced thrombosis of a small vessel caused by microembolism by selectively occluding a PA just before diving into the cortex (radius; 10-25 µm) in the mouse. Clotting was induced with FeCl applied focally over the PA by a glass micropipette for 3 min. DC potential changes were recorded and the alterations in cortical blood flow were monitored by laser speckle contrast imaging. Mice were kept alive for 1-4 weeks and brain sections were stained with H&E or luxol-fast blue to evaluate changes induced by PA occlusion. We found that single PA occlusion consistently triggered a CSD originating from the tissue around the PA soon after occlusion and induced delayed, small ischemic lesions within territory of the affected vessel a few weeks later. These findings suggest that cerebral microembolism can lead to MA attacks and may account for some of the silent brain lesions.
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http://dx.doi.org/10.1016/j.brainres.2017.11.023DOI Listing
January 2018

Angiographic Microcirculatory Obstructions Distal to Occlusion Signify Poor Outcome after Endovascular Treatment for Acute Ischemic Stroke.

Transl Stroke Res 2018 02 19;9(1):44-50. Epub 2017 Aug 19.

Department of Neurology, Faculty of Medicine, and Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

The success of endovascular therapies depends on a number of factors including flow dynamics proximal and distal to the occlusion. The evaluation of antegrade flow distal to the occluded segment is currently a readily available, yet unexplored, option during stentriever-mediated thrombectomy. In this study, we retrospectively evaluated presence of contrast stasis and absence of capillary blush on angiograms obtained by selective injections into the distal site of occlusive thrombi, prior to deployment of stentrievers, in patients undergoing endovascular treatment for acute MCA occlusion. The role of this novel angiographic strategy assessing distal antegrade flow in predicting procedural and clinical outcome was compared to previously defined, prognostic angiographic characteristics. A total of 7 (21%) out of 34 patients had contrast stasis and lack of capillary blush downstream to the injection site. None of these patients with angiographic features suggestive of microcirculatory obstructions achieved a satisfactory outcome in terms of reperfusion (TICI grade 2B-C) and clinical outcome (90-day mRS 0-2), while the corresponding figures were 78% (p < 0.001) and 48% (p = 0.029), respectively, among patients with substantial capillary filling. These findings highlight the potentially detrimental impact of impaired microcirculatory flow on tissue and clinical outcome after recanalization/reperfusion efforts in acute ischemic stroke.
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http://dx.doi.org/10.1007/s12975-017-0562-2DOI Listing
February 2018

Systemically administered brain-targeted nanoparticles transport peptides across the blood-brain barrier and provide neuroprotection.

J Cereb Blood Flow Metab 2015 Mar 10;35(3):469-75. Epub 2014 Dec 10.

Department of Neurology, Faculty of Medicine, Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Although growth factors and anti-apoptotic peptides have been shown to be neuroprotective in stroke models, translation of these experimental findings to clinic is hampered by limited penetration of peptides to the brain. Here, we show that a large peptide like the basic fibroblast growth factor (bFGF) and a small peptide inhibitor of caspase-3 (z-DEVD-FMK) can effectively be transported to the brain after systemic administration by incorporating these peptides to brain-targeted nanoparticles (NPs). Chitosan NPs were loaded with peptides and then functionalized by conjugating with antibodies directed against the transferrin receptor-1 on brain endothelia to induce receptor-mediated transcytosis across the blood-brain barrier (BBB). Pre-ischemic systemic administration of bFGF- or z-DEVD-FMK-loaded NPs significantly decreased the infarct volume after 2-hour middle cerebral artery occlusion and 22-hour reperfusion in mice. Co-administration of bFGF- or z-DEVD-FMK-loaded NPs reduced the infarct volume further and provided a 3-hour therapeutic window. bFGF-loaded NPs were histologically detected in the brain parenchyma and also restored ischemia-induced Akt dephosphorylation. The neuroprotection was not observed when receptor-mediated transcytosis was inhibited with imatinib or when bFGF-loaded NPs were not conjugated with the targeting antibody, which enables them to cross the BBB. Nanoparticles targeted to brain are promising drug carriers to transport large as well as small BBB-impermeable therapeutics for neuroprotection against stroke.
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http://dx.doi.org/10.1038/jcbfm.2014.220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348388PMC
March 2015

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury.

Nat Nanotechnol 2014 24;9(12):1054-1062. Epub 2014 Nov 24.

1] Institut Galien Paris-Sud UMR CNRS 8612, Faculty of Pharmacy, University of Paris-Sud XI, 92296 Châtenay-Malabry, France [2] Labex d'Excellence NanoSaclay.

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.
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http://dx.doi.org/10.1038/nnano.2014.274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351925PMC
November 2014

Microvascular protection is essential for successful neuroprotection in stroke.

J Neurochem 2012 Nov;123 Suppl 2:2-11

Department of Neurology, Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Currently, the best way of neuroprotection for acute ischemic stroke appears to be restoration of blood flow to the ischemic area by thrombolysis. Unfortunately, a short therapeutic time window as well as thrombolysis-induced bleeding and edema limit the use of recanalization therapies. Here, we review the evidence suggesting that ischemia/reperfusion-induced microvascular injury plays a critical role in determining tissue survival after recanalization in focal cerebral ischemia by disrupting the blood-brain barrier integrity and promoting microcirculatory clogging. Among many complex mechanisms of the ischemia-reperfusion injury, overproduction of oxygen and nitrogen radicals on the microvascular wall appears to significantly contribute to these pathological processes. These developments bring about the exciting possibility that effective suppression of oxidative/nitrative stress during pharmacological or interventional re-opening of the occluded artery may significantly improve the outcome of recanalization therapies in stroke patients by improving microcirculatory reflow as well as by preventing hemorrhagic conversion and vasogenic edema. They also point to the critical (but partly neglected) importance of the microcirculation in neuroprotection.
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http://dx.doi.org/10.1111/j.1471-4159.2012.07938.xDOI Listing
November 2012

Transport of a caspase inhibitor across the blood-brain barrier by chitosan nanoparticles.

Methods Enzymol 2012 ;508:253-69

Institute of Neurological Sciences and Psychiatry and, Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.

The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Several approaches taking advantage of molecular and physicochemical characteristics of the BBB have been developed recently to improve drug delivery to the brain. Here, we introduce a nanomedicine that can efficiently transport BBB-impermeable peptides to the brain. This nanomedicine is made of chitosan nanoparticles into which considerable amounts of a peptide can be incorporated. The nanoparticle surface is modified with polyethylene glycol to enhance the plasma residence time by preventing their capture by the reticuloendothelial system. Monoclonal antibodies against the transferrin receptor (TfR), which is highly expressed on the brain capillary endothelium, are conjugated to nanoparticles via biotin-streptavidin bonds. The activation of TfR by the nanoparticle-antibody complex induces transcytosis and thus delivers the loaded drug to the brain. Penetration of nanoparticles to the brain can be illustrated in vivo by intravital microscopy as well as ex vivo by fluorescence or electron microscopy. N-Benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK)-loaded nanoparticles rapidly release their contents within brain parenchyma, inhibit ischemia-induced caspase-3 activity, and thereby provide neuroprotection.
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http://dx.doi.org/10.1016/B978-0-12-391860-4.00013-6DOI Listing
July 2012

Brain microvascular pericytes in health and disease.

Acta Neuropathol 2011 Jul 9;122(1):1-9. Epub 2011 Jun 9.

Department of Neurology, Faculty of Medicine, Institute of Neurological Sciences and Psychiatry, Hacettepe University, Sihhiye, Ankara, Turkey.

Pericytes are located at periphery of the microvessel wall and wrap it with their processes. They communicate with other cells of the neurovascular unit by direct contact or through signaling pathways and regulate several important microcirculatory functions. These include development and maintenance of the blood-brain barrier (BBB), distribution of the capillary blood flow to match the local metabolic need of the nearby cells, and angiogenesis. Pericytes also exhibit phagocytic activity and may function as pluripotent stem cells. Increasing evidence suggests a role for pericytes in a wide range of CNS diseases. They appear to be vulnerable to oxygen and nitrogen radical toxicity and have been shown to contract during cerebral ischemia and remain contracted despite reopening of the occluded artery. This causes impaired re-flow and may diminish the benefit of re-canalization therapies in stroke patients. Hyperglycemia-induced dysfunction of the signaling pathways between pericytes and endothelia is thought to play an important role in diabetic retinopathy, a common cause of blindness. Amyloid deposits detected within degenerating pericytes in the brains of patients with Alzheimer's disease suggest that pericyte dysfunction may play a role in cerebral hypoperfusion and impaired amyloid β-peptide clearance in Alzheimer's disease. This exciting possibility may reveal a novel temporal sequence of events in chronic neurodegeneration, in which microvascular dysfunction due to pericyte degeneration initiates secondary neurodegenerative changes. Identification of molecular mechanisms by which pericytes regulate BBB integrity in inflammatory conditions as well as in vasogenic brain edema may lead to new treatments. Pericytes may also take part in tissue repair and vascularization after CNS injury. In conclusion, although the evidence is just emerging and mostly preliminary, disclosing pericytes' role in the pathophysiology of CNS diseases may yield exciting developments and novel treatments.
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http://dx.doi.org/10.1007/s00401-011-0847-6DOI Listing
July 2011

Fingolimod provides long-term protection in rodent models of cerebral ischemia.

Ann Neurol 2011 Jan 12;69(1):119-29. Epub 2010 Nov 12.

Department of Radiology, Massachusetts General Hospital, Charlestown, MA, USA.

Objective: The sphingosine-1-phosphate (S1P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species.

Methods: We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod.

Results: In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by tumor necrosis factor alpha.

Interpretation: These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.
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http://dx.doi.org/10.1002/ana.22186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3200194PMC
January 2011

Alpha-synuclein aggregation induced by brief ischemia negatively impacts neuronal survival in vivo: a study in [A30P]alpha-synuclein transgenic mouse.

J Cereb Blood Flow Metab 2011 Mar 29;31(3):913-23. Epub 2010 Sep 29.

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.

Alpha-synuclein oligomerization and aggregation are considered to have a role in the pathogenesis of neurodegenerative diseases. However, despite numerous in vitro studies, the impact of aggregates in the intact brain is unclear. In vitro, oxidative/nitrative stress and acidity induce α-synuclein oligomerization. These conditions favoring α-synuclein fibrillization are present in the ischemic brain, which may serve as an in vivo model to study α-synuclein aggregation. In this study, we show that 30-minute proximal middle cerebral artery (MCA) occlusion and 72 hours reperfusion induce oligomerization of wild-type α-synuclein in the ischemic mouse brain. The nonamyloidogenic isoform β-synuclein did not form oligomers. Alpha-synuclein aggregates were confined to neurons and colocalized with ubiquitin immunoreactivity. We also found that 30 minutes proximal MCA occlusion and 24 hours reperfusion induced larger infarcts in C57BL/6(Thy1)-h[A30P]alphaSYN transgenic mice, which have an increased tendency to form synuclein fibrils. Trangenics also developed more selective neuronal necrosis when subjected to 20 minutes distal MCA occlusion and 72 hours reperfusion. Enhanced 3-nitrotyrosine immunoreactivity in transgenic mice suggests that oxidative/nitrative stress may be one of the mechanisms mediating aggregate toxicity. Thus, the increased vulnerability of transgenic mice to ischemia suggests that α-synuclein aggregates not only form during ischemia but also negatively impact neuronal survival, supporting the idea that α-synuclein misfolding may be neurotoxic.
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http://dx.doi.org/10.1038/jcbfm.2010.170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063624PMC
March 2011

A nanomedicine transports a peptide caspase-3 inhibitor across the blood-brain barrier and provides neuroprotection.

J Neurosci 2009 Nov;29(44):13761-9

Department of Neurology, Faculty of Medicine and Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Caspases play an important role as mediators of cell death in acute and chronic neurological disorders. Although peptide inhibitors of caspases provide neuroprotection, they have to be administered intracerebroventricularly because they cannot cross the blood-brain barrier (BBB). Herein, we present a nanocarrier system that can transfer chitosan nanospheres loaded with N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK), a relatively specific caspase-3 inhibitor, across BBB. Caspase-3 was chosen as a pharmacological target because of its central role in cell death. Polyethylene glycol-coated nanospheres were conjugated to an anti-mouse transferrin receptor monoclonal antibody (TfRMAb) that selectively recognizes the TfR type 1 on the cerebral vasculature. We demonstrate with intravital microscopy that this nanomedicine is rapidly transported across the BBB without being measurably taken up by liver and spleen. Pre- or post-treatment (2 h) with intravenously injected Z-DEVD-FMK-loaded nanospheres dose dependently decreased the infarct volume, neurological deficit, and ischemia-induced caspase-3 activity in mice subjected to 2 h of MCA occlusion and 24 h of reperfusion, suggesting that they released an amount of peptide sufficient to inhibit caspase activity. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17 after closure of the BBB. Neither nanospheres functionalized with TfRMAb but not loaded with Z-DEVD-FMK nor nanospheres lacking TfRMAb but loaded with Z-DEVD-FMK had any effect on either paradigm, suggesting that inhibition of caspase activity and subsequent neuroprotection were due to efficient penetration of the peptide into brain. Thus, chitosan nanospheres open new and exciting opportunities for brain delivery of biologically active peptides that are useful for the treatment of CNS disorders.
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http://dx.doi.org/10.1523/JNEUROSCI.4246-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666719PMC
November 2009

Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery.

Nat Med 2009 Sep 30;15(9):1031-7. Epub 2009 Aug 30.

Department of Neurology, Faculty of Medicine and Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Here we show that ischemia induces sustained contraction of pericytes on microvessels in the intact mouse brain. Pericytes remain contracted despite successful reopening of the middle cerebral artery after 2 h of ischemia. Pericyte contraction causes capillary constriction and obstructs erythrocyte flow. Suppression of oxidative-nitrative stress relieves pericyte contraction, reduces erythrocyte entrapment and restores microvascular patency; hence, tissue survival improves. In contrast, peroxynitrite application causes pericyte contraction. We also show that the microvessel wall is the major source of oxygen and nitrogen radicals causing ischemia and reperfusion-induced microvascular dysfunction. These findings point to a major but previously not recognized pathophysiological mechanism; ischemia and reperfusion-induced injury to pericytes may impair microcirculatory reflow and negatively affect survival by limiting substrate and drug delivery to tissue already under metabolic stress, despite recanalization of an occluded artery. Agents that can restore pericyte dysfunction and microvascular patency may increase the success of thrombolytic and neuroprotective treatments.
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http://dx.doi.org/10.1038/nm.2022DOI Listing
September 2009

Protective role of 27bp repeat polymorphism in intron 4 of eNOS gene in lacunar infarction.

Free Radic Res 2009 Mar 31;43(3):272-9. Epub 2009 Jan 31.

Department of Neurology, Hacettepe University, Ankara, Turkey.

Association of the three potential endothelial nitric oxide synthase gene (eNOS) polymorphisms (T-786C in promoter region, G894T in exon 7 and tandem 27-bp repeats in intron 4) with an increased risk of lacunar infarction (LI) were investigated. Genotypes of 70 patients and 81 healthy controls were determined through PCR with or without RFLP. Flow-mediated dilatation (FMD) was performed to assess endothelial-dependent vasodilatation, whereas the endothelial-independent vasodilatation was assessed with nitroglycerin (NTG). Genotype distribution was significantly different between LI patients and controls for intron 4aa (alleles for four repeats), genotype frequency being 1.4% and 16.0%, respectively (odds ratio for additive effect, 0.47; 95% CI, 0.28-0.81; p=0.006). Haplotypes with the intron 4aa polymorphism were significantly higher in controls when compared with the LI group (p=0.001). Diminished FMD but normal NTG response confirmed that patients with LI have generalized endothelial dysfunction. Intron 4aa genotype of eNOS gene seems to be protective for isolated LI and the effect was potentiated by the absence of 786C polymorphism in any allele of the promoter region.
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http://dx.doi.org/10.1080/10715760802691489DOI Listing
March 2009

Hydrogen peroxide-induced stroke: elucidation of the mechanism in vivo.

J Neurosurg 2009 Jan;110(1):94-100

Department of Neurosurgery, Hacettepe University, Ankara, Turkey.

Object: Hydrogen peroxide (H2O2) is used as a hemostatic agent in many neurosurgery centers. The authors used a 3% H2O2 solution for final hemostasis after removal of a left insular tumor. Immediately afterward, air bubbles were observed within the lumen of the polar temporal artery. Postoperative MR imaging revealed punctate areas of infarction in the lenticulostriate artery territory. The authors designed an experimental study to elucidate the mechanism of remote O2 emboli and reactive O2 species-related vasoactive responses and thrombus formation.

Methods: In this study, H2O2 irrigation was used in mice with either an intact pial layer or after the pia mater was removed through a corticotomy. Normal saline irrigation was used in the corresponding control groups. Vessels were examined for intravascular O2 emboli under the microscope. Tissue sections were then obtained and stained with H & E and the 3-nitrotyrosine (3-NT) antibody to evaluate intravascular thrombus formation and peroxynitrite reaction, respectively.

Results: Multiple bubbles were observed within the lumen of the vessels after exposure to H2O2 regardless of whether the pial layer was destroyed or intact. Immunofluorescent staining for 3-NT showed an abundant positive reaction in the vessel walls of all animals exposed to H2O2 as well as vascular occlusion with acute thrombus formation. Samples taken from the animals that received saline showed no positive staining for 3-NT and no vascular occlusion.

Conclusions: Exposure to H2O2 may cause serious ischemic complications. The formation of peroxynitrite may cause vasoactive responses to H2O2 and platelet aggregation/thrombus formation, and the free diffusion of H2O2 through the vessel walls and its conversion to water and O2 leads to O2 bubbles within the closed vessel lumen. If used intradurally, H2O2 may have deleterious ischemic effects, and it can only be used carefully in open extradural spaces.
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http://dx.doi.org/10.3171/2008.3.17434DOI Listing
January 2009

Statin potentiates human platelet eNOS activity without enhancing eNOS mRNA and protein levels.

Cerebrovasc Dis 2008 15;26(2):190-8. Epub 2008 Jul 15.

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.

Background/aims: Experimental studies suggest an enhanced endothelial and platelet nitric oxide (NO) generation after statin treatment, possibly due to increased endothelial NO synthase (eNOS) activity and protein levels. In parallel with experimental research, statins were shown to increase the forearm blood flow independently of serum cholesterol in humans. However, it was not possible to correlate blood flow changes with eNOS levels in these studies due to limitations in obtaining arterial samples. Hence, we investigated changes in eNOS activity, mRNA and protein levels after statin treatment in human platelets, which are readily accessible unlike arteries.

Methods: In vitro bleeding times were measured in 22 patients by stimulating platelets with collagen-epinephrine or collagen-ADP. To assess platelet eNOS activity, the bleeding times were also determined after incubating platelets with L-arginine. The measurements were repeated following 14 days of pravastatin (40 mg/day) treatment. Platelet-rich plasma was collected before and after statin treatment to evaluate eNOS mRNA (semiquantitative RT-PCR) and protein levels (Western blotting).

Results: The basal bleeding time was prolonged by 24 +/- 3% (mean +/- SE) when the samples were incubated with 500 microM of L-arginine. The NOS inhibitor L-N(5)-(I-iminoethyl)ornithine reversed this effect, suggesting that it was mediated by NO. After statin treatment, the NO-mediated prolongation of the bleeding time with 500 microM of L-arginine was significantly potentiated (to 44 +/- 10%). Despite enhanced eNOS activity, there was no significant change in platelet eNOS mRNA and protein levels after statin treatment.

Conclusion: These data demonstrate that platelet eNOS activity is potentiated after statin treatment in humans in parallel with experimental studies.
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http://dx.doi.org/10.1159/000145327DOI Listing
October 2008
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