MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

Electrical Stimulation of the Mesencephalic Locomotor Region Has No Impact on Blood-Brain Barrier Alterations after Cerebral Photothrombosis in Rats.

Blood-brain barrier (BBB) disruption is a critical event after ischemic stroke, which results in edema formation and hemorrhagic transformation of infarcted tissue. BBB dysfunction following stroke is partly mediated by proinflammatory agents. We recently have shown that high frequency stimulation of the mesencephalic locomotor region (MLR-HFS) exerts an antiapoptotic and anti-inflammatory effect in the border zone of cerebral photothrombotic stroke in rats. Whether MLR-HFS also has an impact on BBB dysfunction in the early stage of stroke is unknown. In this study, rats were subjected to photothrombotic stroke of the sensorimotor cortex and implantation of a stimulating microelectrode into the ipsilesional MLR. Thereafter, either HFS or sham stimulation of the MLR was applied for 24 h. After scarifying the rats, BBB disruption was assessed by determining albumin extravasation and tight junction integrity (claudin 3, claudin 5, and occludin) using Western blot analyses and immunohistochemistry. In addition, by applying zymography, expression of pro-metalloproteinase-9 (pro-MMP-9) was analyzed. No differences were found regarding infarct size and BBB dysfunction between stimulated and unstimulated animals 24 h after induction of stroke. Our results indicate that MLR-HFS neither improves nor worsens the damaged BBB after stroke. Attenuating cytokines/chemokines in the perilesional area, as mediated by MLR-HFS, tend to play a less significant role in preventing the BBB integrity.

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats.

BACKGROUND AND PURPOSE: Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. METHODS: For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. RESULTS: Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. CONCLUSIONS: With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.

Imaging of the peripheral nervous system.

This chapter summarizes progress in the evaluation of peripheral nerve (PN) lesions and disorders by imaging techniques encompassing magnetic resonance imaging (MRI) and nerve ultrasound (US). Due to the radiation exposure and limited sensitivity in soft tissue contrast, computed-tomography (CT) plays no significant role in the diagnostic work-up of PN disorders. MRI and US are complementary techniques for the evaluation of peripheral nerves, each having particular advantages and disadvantages. Nerve injury induces intrinsic MRI signal alterations on T2-weighted sequences in degenerating or demyelinating nerve segments as well as in corresponding muscle groups exhibiting denervation which can be exploited diagnostically. Nerve US is based on changes in the nerve echotexture due to tumor formation or focal enlargement caused by entrapment or inflammation. Both MRI and US provide morphological information on the precise site and extent of nerve injury. While US has the advantage of easy accessibility, providing images with superior spatial resolution at low cost, MRI shows better soft tissue contrast and better image quality for deep-lying nerve structures since imaging is not hindered by bone. Recent advances have remarkably increased spatial resolution of both MRI and US making imaging indispensible for the elucidation of causes of nerve compression, peripheral nerve tumors, and focal inflammatory conditions. Both MRI and US further guide neurosurgical exploration and can simplify treatment. Importantly, imaging can reveal treatable conditions even in the absence of gross electrophysiological alterations, illustrating its increasing role in clinical practice. In experimental settings, novel molecular and cellular MRI contrast agents allow in-vivo assessment of nerve regeneration as well as monitoring of neuroinflammation. Depending on further clinical development, contrast-enhanced MRI has the potential to follow cellular responses over time in vivo and to overcome the current limitations of histological assessment of nerve afflictions. Further advances in contrast-enhanced US has the potential for developing into a tool for the assessment of nerve blood perfusion, paving the way for better assessments of ischemic neuropathies.

The neuroprotective impact of the leak potassium channel TASK1 on stroke development in mice.

Oxygen depletion (O(2)) and a decrease in pH are initial pathophysiological events in stroke development, but secondary mechanisms of ischemic cell death are incompletely understood. By patch-clamp recordings of brain slice preparations we show that TASK1 and TASK3 channels are inhibited by pH-reduction (42+/-2%) and O(2) deprivation (36+/-5%) leading to membrane depolarization, increased input resistance and a switch in action potential generation under ischemic conditions. In vivo TASK blockade by anandamide significantly increased infarct volumes at 24 h in mice undergoing 30 min of transient middle cerebral artery occlusion (tMCAO). Moreover, blockade of TASK channels accelerated stroke development. Supporting these findings TASK1(-/-) mice developed significantly larger infarct volumes after tMCAO accompanied by worse outcome in functional neurological tests compared to wild type mice. In conclusion, our data provide evidence for an important role of functional TASK channels in limiting tissue damage during cerebral ischemia.

Technology insight: visualizing peripheral nerve injury using MRI.

Currently, the evaluation of peripheral nerve disorders depends on clinical examination, supplemented by electrophysiological studies. These approaches provide general information on the distribution and classification of nerve lesions-for example, axonal versus demyelinative-but nerve biopsies are still required to obtain morphological and pathophysiological details. In this article, we review recent progress in the imaging of peripheral nerve injury by magnetic resonance (MR) neurography. Axonal nerve injury leads to Wallerian degeneration, resulting in a hyperintense nerve signal on T2-weighted MR images of the distal nerve segment. This signal is lost following successful regeneration. Concomitant denervation-induced signal alterations in muscles can further help us to determine whether nerve trunks or roots are affected. These signal changes are caused by various combinations of nonspecific tissue alterations, however, and are not related to particular pathoanatomical findings, such as inflammation, demyelination or axonal injury. New experimental MR contrast agents, such as gadofluorine M and superparamagnetic iron oxide particles, allow visualization of the dynamics of peripheral nerve injury and repair. Further clinical development of these MR contrast agents should allow these functional aspects of nerve injury and repair to be assessed in humans, thereby aiding the differential diagnosis of peripheral nerve disorders.

Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.

In this study, we adapted the original rat photothrombosis model of Watson et al. (Ann Neurol 17 (1985) 497) for use in mice by refining the application route of the dye, illumination and stereotactic parameters. After intraperitoneal injection of the photosensitive dye Rose bengal, subsequent focal illumination of the brain with a cold light source through the intact skull led to focal cortical infarcts of reproducible size, location and geometry. Cresyl violet histology displayed well-demarcated infarcts that matured with time in a predictable manner. Microglial responses, as assessed by immunocytochemistry, against F4/80 and CD11b antigens were rapid and complete at the infarct site, but delayed and incomplete in degenerating fiber tracts and ipsilateral thalamic nuclei. In contrast to the rat, where the expression of CD4 and CD8 antigens discriminate distinct subpopulations of lesion-associated phagocytes, the expression of both markers was low to absent in the mouse model. In both rats and mice, cerebral photothrombosis shares essential inflammatory responses with focal ischemia induced by middle cerebral artery occlusion. It may provide a useful model to study functional aspects of lesion-associated and remote molecular responses in transgenic mice.