Hulya Karatas

Spreading Depression Triggers Headache by Activating Neuronal Panx1 Channels

How Migraine Develops Migraine is a common medical disorder. Unfortunately, how and why migraine headache is initiated is unclear. Karatas et al. (p. 1092) now describe a signaling pathway between stressed neurons and meningeal trigeminal afferents, which may explain how migraine headaches can be generated. Migraine results from a sequence of events starting from stressed cortical neurons and leading to the trigeminal nucleus. The initial phase in the development of a migraine is still poorly understood. Here, we describe a previously unknown signaling pathway between stressed neurons and trigeminal afferents during cortical spreading depression (CSD), the putative cause of migraine aura and headache. CSD caused neuronal Pannexin1 (Panx1) megachannel opening and caspase-1 activation followed by high-mobility group box 1 (HMGB1) release from neurons and nuclear factor κB activation in astrocytes. Suppression of this cascade abolished CSD-induced trigeminovascular activation, dural mast cell degranulation, and headache. CSD-induced neuronal megachannel opening may promote sustained activation of trigeminal afferents via parenchymal inflammatory cascades reaching glia limitans. This pathway may function to alarm an organism with headache when neurons are stressed.

A Nanomedicine Transports a Peptide Caspase-3 Inhibitor across the Blood–Brain Barrier and Provides Neuroprotection

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.

Inhibition of 12/15-lipoxygenase as therapeutic strategy to treat stroke.

Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15‐lipoxygenase was increased in the ischemic mouse brain, and 12/15‐lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis‐inducing factor. A novel inhibitor of 12/15‐lipoxygenase, LOXBlock‐1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock‐1 was protective. Furthermore, it reduced tissue plasminogen activator‐associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15‐lipoxygenase as a viable strategy for first‐line stroke treatment. Ann Neurol 2013

Investigation of HSV-1, HSV-2, CMV, HHV-6 and HHV-8 DNA by real-time PCR in surgical resection materials of epilepsy patients with mesial temporal lobe sclerosis

OBJECTIVE The objective of this study is to investigate the presence of viral DNAs of HSV-1, HSV-2, HHV-6, HHV-8, and CMV in hippocampus of the patients with mesial temporal lobe epilepsy (MTLE) syndrome. METHODS Pathological specimens were obtained from 33 patients with MTLE undergone temporal lobectomy with amygdalo-hippocampectomy due to intractable seizures. Autopsy materials from the hippocampus of 7 patients without neurological disease were used as controls. The data was also correlated with the clinical history of patients including febrile convulsions, age, and history of CNS infections. Real-time polymerase chain reaction method was performed for detection of DNAs of these viruses. RESULTS HHV-6, HSV-1 and HHV-8 were detected in the hippocampus of 3, 2 and 1 patients with MTLE respectively. None of the hippocampus of patients with MTLE was positive for DNA of HSV-2 and/or CMV. Three patients with positive HHV-6 DNAs had febrile convulsions and family history for epilepsy. None of our control specimens showed PCR positivity to any of the 5 tested viruses. CONCLUSIONS Our study is the first to report the presence of HHV-8 viral genome in the brain tissue of patient with MTLE. Viral DNAs were detected in a total of 18% of the patients in this study; we can conclude that activity of the latent virus in patients with hippocampal sclerosis should be more extensively studied to establish its role in active infection.

Thrombotic distal middle cerebral artery occlusion produced by topical FeCl3 application: a novel model suitable for intravital microscopy and thrombolysis studies

Intravital or multiphoton microscopy and laser-speckle imaging have become popular because they allow live monitoring of several processes during cerebral ischemia. Available rodent models have limitations for these experiments; e.g., filament occlusion of the proximal middle cerebral artery (MCA) is difficult to perform under a microscope, whereas distal occlusion methods may damage the MCA and the peri-arterial cortex. We found that placement of a 10% FeCl3-soaked filter paper strip (0.3 × 1 mm2) on the duramater over the trunk of the distal MCA through a cranial window for 3 minutes induced intraarterial thrombus without damaging the peri-arterial cortex in the mouse. This caused a rapid regional cerebral blood flow decrease within 10 minutes and total occlusion of the MCA segment under the filter paper in 17 ± 2 minutes, which resulted in a typical cortical infarct of 27 ±4 mm3 at 24 hours and moderate sensorimotor deficits. There was no significant hemispheric swelling or hemorrhage or mortality at 24 hours. Reperfusion was obtained in half of the mice with tissue plasminogen activator, which allowed live monitoring of clot lysis along with restoration of tissue perfusion and MCA flow. In conclusion, this relatively simple and noninvasive stroke model is easy to perform under a microscope, making it suitable for live imaging and thrombolysis studies.

Involvement of the central nervous system in Miller Fisher syndrome: a case report

Miller Fisher syndrome (MFS) is characterised by ophthalmoplegia, ataxia and areflexia. Reports on cerebellar ataxia and supranuclear oculomotor derangement in MFS suggested an additional involvement of the central nervous system (CNS), resembling Bickerstaffs brainstem encephalitis (BBE). In the present report, a patient with a monophasic acute illness, early recovery and specific clinical-laboratory findings suggested both intrinsic brainstem and peripheral nerve disease (MFS and BBE). In pons and medulla oblangata, blurred to discrete T2-lesions were revealed by cranial MRI, while involvement of peripheral nerves was detected with EMG. The CSF showed no increase in protein or cell content, such as occurs in brainstem encephalitis.

A new model of transient focal cerebral ischemia for inducing selective neuronal necrosis.

Brief cerebral ischemia leads to selective neuronal necrosis (SNN), which is characterized by neuronal death with sparing of glial and vascular elements of the central nervous system. Understanding the pathophysiology of SNN may help elucidating the mechanisms and consequences of neuronal injury in humans following brief ischemia. Contrary to the presence of reproducible models of transient global ischemia, animal models of transient focal ischemia producing SNN are scarce and have important limitations such as causing ischemia in a vast area and inducing additional insults. In this study, we developed a practical mouse model of SNN without these limitations, by compressing the distal middle cerebral artery (MCA) with a blunted micropipette for 15 min. The success of compression was evaluated by monitoring the regional cerebral blood flow, and conventional histopathology and immunolabeling of the brain sections. Seven/fourteen days after ischemia, intracisternally administered propidium iodide labeled numerous necrotic cells in the frontoparietal cortex, which were mostly NeuN-positive, but were not immunolabeled with astrocytic markers (GFAP and S100), and showed neuronal morphology with hematoxylin-eosin staining, indicating that the model successfully induced ischemic injury limited to neurons. The model could become an important tool for investigating the long-term effects of brief ischemic events like transient ischemic attacks and could offer convenient reversible distal MCA occlusion for studies using intravital microscopy.

Imaging of Contrast Medium Extravasation in Anticoagulation-Associated Intracerebral Hemorrhage With Dual-Energy Computed Tomography

Background and Purpose— Contrast medium extravasation (CE) in intracerebral hemorrhage (ICH) is a marker of ongoing bleeding and a predictor of hematoma expansion. The aims of the study were to establish an ICH model in which CE can be quantified, characterized in ICH during warfarin and dabigatran anticoagulation, and to evaluate effects of prothrombin complex concentrates on CE in warfarin-associated ICH. Methods— CD1-mice were pretreated orally with warfarin, dabigatran, or vehicle. Prothrombin complex concentrates were administered in a subgroup of warfarin-treated mice. ICH was induced by stereotactic injection of collagenase VIIs into the right striatum. Contrast agent (350 &mgr;L Isovue 370 mg/mL) was injected intravenously after ICH induction (2–3.5 hours). Thirty minutes later, mice were euthanized, and CE was measured by quantifying the iodine content in the hematoma using dual-energy computed tomography. Results— The optimal time point for contrast injection was found to be 3 hours after ICH induction, allowing detection of both an increase and a decrease of CE using dual-energy computed tomography. CE was higher in the warfarin group compared with the controls (P=0.002). There was no significant difference in CE between dabigatran-treated mice and controls. CE was higher in the sham-treated warfarin group than in the prothrombin complex concentrates–treated warfarin group (P<0.001). Conclusions— Dual-energy computed tomography allows quantifying CE, as a marker of ongoing bleeding, in a model of anticoagulation-associated ICH. Dabigatran induces less CE in ICH than warfarin and consequently reduces risks of hematoma expansion. This constitutes a potential safety advantage of dabigatran over warfarin. Nevertheless, in case of warfarin anticoagulation, prothrombin complex concentrates reduce this side effect.

STAT-Dependent Upregulation of 12/15-Lipoxygenase Contributes to Neuronal Injury after Stroke

Oxidative stress is a major brain injury mechanism after ischemic stroke. 12/15-lipoxygenase (12/15-LOX) is a key mediator of oxidative stress, contributing to neuronal cell death and vascular leakage. Nonetheless, the mechanism leading to its upregulation is currently unknown. We show here that Signal Transducers and Activators of Transcription (STATs), specifically STAT6 and possibly STAT1, increase transcription of 12/15-LOX in neuronal cells. Both p-STAT6 and −1 bound to specific STAT binding sites in the mouse 12/15-LOX promoter. Small interfering RNA (siRNA) knockdown showed STAT6 to be the dominant regulator, reducing 12/15-LOX promoter activation and cell death in oxidatively stressed HT22 cells. STAT6 siRNA efficiently prevented the increase of 12/15-LOX in murine primary neurons, both after induction of oxidative stress and after oxygen-glucose deprivation. Early activation of STAT6 and STAT1 in mice was consistent with a role in regulating 12/15-LOX in focal ischemia. Brains of human stroke patients showed increased p-STAT6 and p-STAT1 in the peri-infarct region, along with 12/15-LOX and markers of apoptosis. These results link STAT6 and STAT1 to the 12/15-LOX damage pathway and suggest disregulation of STAT-dependent transcription as injury mechanism in stroke. Selectively targeting STATs may thus be a novel therapeutic approach to reducing brain injury after a stroke.

Changes in the Expression of Selenoproteins in Mesial Temporal Lobe Epilepsy Patients

Selenoproteins are enzymes containing selenium in their structure and are involved in cellular processes such as defense against oxidative stress and cell survival. The aim of this study is to investigate the expression of four selenoproteins (GPX1, TRXR1, SELP and SELW) in the hippocampus of intractable mesial temporal lobe epilepsy (MTLE) patients who underwent curative surgery. The selenoproteins is investigated at the mRNA level via RT-PCR and in situ hybridization and by immunostaining at the protein level. The expression of SELW exhibited a relative induction of more than tenfold, and immunostaining findings provided evidence that this upregulation is confined to neurons. GPX1 was also upregulated 2.3-fold, and TRXR1 was downregulated between 70 and 20% in MTLE patients. The profound induction of SELW has been accompanied by GPX1 and displayed a strong correlation with BCL2 expression, suggesting a protective role for these selenoproteins, and may be an indicator of a defense mechanism in surviving neurons.