Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes.

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an "inflammatory signaling cascade" in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

Cortical spreading depression can be triggered by sensory stimulation in primed wild type mouse brain: a mechanistic insight to migraine aura generation.

BACKGROUND: Unlike the spontaneously appearing aura in migraineurs, experimentally, cortical spreading depression (CSD), the neurophysiological correlate of aura is induced by non-physiological stimuli. Consequently, neural mechanisms involved in spontaneous CSD generation, which may provide insight into how migraine starts in an otherwise healthy brain, remain largely unclear. We hypothesized that CSD can be physiologically induced by sensory stimulation in primed mouse brain. METHODS: Cortex was made susceptible to CSD with partial inhibition of Na+/K+-ATPase by epidural application of a low concentration of Na+/K+-ATPase blocker ouabain, allowing longer than 30-min intervals between CSDs or by knocking-down α2 subunit of Na+/K+-ATPase, which is crucial for K+ and glutamate re-uptake, with shRNA. Stimulation-triggered CSDs and extracellular K+ changes were monitored in vivo electrophysiologically and a K+-sensitive fluoroprobe (IPG-4), respectively. RESULTS: After priming with ouabain, photic stimulation significantly increased the CSD incidence compared with non-stimulated animals (44.0 vs. 4.9%, p < 0.001). Whisker stimulation also significantly increased the CSD incidence, albeit less effectively (14.9 vs. 2.4%, p = 0.02). Knocking-down Na+/K+-ATPase (50% decrease in mRNA) lowered the CSD threshold in all mice tested with KCl but triggered CSDs in 14.3% and 16.7% of mice with photic and whisker stimulation, respectively. Confirming Na+/K+-ATPase hypofunction, extracellular K+ significantly rose during sensory stimulation after ouabain or shRNA treatment unlike controls. In line with the higher CSD susceptibility observed, K+ rise was more prominent after ouabain. To gain insight to preventive mechanisms reducing the probability of stimulus-evoked CSDs, we applied an A1-receptor antagonist (DPCPX) to the occipital cortex, because adenosine formed during stimulation from ATP can reduce CSD susceptibility. DPCPX induced spontaneous CSDs but only small-DC shifts along with suppression of EEG spikes during photic stimulation, suggesting that the inhibition co-activated with sensory stimulation could limit CSD ignition when K+ uptake was not sufficiently suppressed as with ouabain. CONCLUSIONS: Normal brain is well protected against CSD generation. For CSD to be ignited under physiological conditions, priming and predisposing factors are required as seen in migraine patients. Intense sensory stimulation has potential to trigger CSD when co-existing conditions bring extracellular K+ and glutamate concentrations over CSD-ignition threshold and stimulation-evoked inhibitory mechanisms are overcome.

Inadequate brain glycogen or sleep increases spreading depression susceptibility.

OBJECTIVE: Glycogen in astrocyte processes contributes to maintenance of low extracellular glutamate and K+ concentrations around excitatory synapses. Sleep deprivation (SD), a common migraine trigger, induces transcriptional changes in astrocytes, reducing glycogen breakdown. We hypothesize that when glycogen utilization cannot match synaptic energy demand, extracellular K+ can rise to levels that activate neuronal pannexin-1 channels and downstream inflammatory pathway, which might be one of the mechanisms initiating migraine headaches. METHODS: We suppressed glycogen breakdown by inhibiting glycogen phosphorylation with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and by SD. RESULTS: DAB caused neuronal pannexin-1 large pore opening and activation of the downstream inflammatory pathway as shown by procaspase-1 cleavage and HMGB1 release from neurons. Six-hour SD induced pannexin-1 mRNA. DAB and SD also lowered the cortical spreading depression (CSD) induction threshold, which was reversed by glucose or lactate supplement, suggesting that glycogen-derived energy substrates are needed to prevent CSD generation. Supporting this, knocking down the neuronal lactate transporter MCT2 with an antisense oligonucleotide or inhibiting glucose transport from vessels to astrocytes with intracerebroventricularly delivered phloretin reduced the CSD threshold. In vivo recordings with a K+ -sensitive/selective fluoroprobe, Asante Potassium Green-4, revealed that DAB treatment or SD caused a significant rise in extracellular K+ during whisker stimulation, illustrating the critical role of glycogen in extracellular K+ clearance. INTERPRETATION: Synaptic metabolic stress caused by insufficient glycogen-derived energy substrate supply can activate neuronal pannexin-1 channels as well as lower the CSD threshold. Therefore, conditions that limit energy supply to synapses (eg, SD) may predispose to migraine attacks, as suggested by genetic studies associating glucose or lactate transporter deficiency with migraine. Ann Neurol 2018;83:61-73.

Treatment of plasminogen deficiency patients with fresh frozen plasma.

Congenital plasminogen (Plg) deficiency leads to the development of ligneous membranes on mucosal surfaces. Here, we report our experience with local and intravenous fresh frozen plasma (FFP). We retrospectively reviewed medical files of 17 patients and their eight first-degree relatives. Conjunctivitis was the main complaint. Thirteen patients were treated both with intravenous and conjunctival FFP. Venous thrombosis did not develop in any. Genetic evaluation revealed heterogeneous mutations as well as polymorphisms. Diagnosis and treatment of Plg deficiency is challenging; topical and intravenous FFP may be an alternative treatment.

Cortical spreading depolarization-induced constriction of penetrating arteries can cause watershed ischemia: A potential mechanism for white matter lesions.

Periventricular white matter lesions (WMLs) are common MRI findings in migraine with aura (MA). Although hemodynamic disadvantages of vascular supply to this region create vulnerability, the pathophysiological mechanisms causing WMLs are unclear. We hypothesize that prolonged oligemia, a consequence of cortical spreading depolarization (CSD) underlying migraine aura, may lead to ischemia/hypoxia at hemodynamically vulnerable watershed zones fed by long penetrating arteries (PAs). For this, we subjected mice to KCl-triggered single or multiple CSDs. We found that post-CSD oligemia was significantly deeper at medial compared to lateral cortical areas, which induced ischemic/hypoxic changes at watershed areas between the MCA/ACA, PCA/anterior choroidal and at the tip of superficial and deep PAs, as detected by histological and MRI examination of brains 2-4 weeks after CSD. BALB-C mice, in which MCA occlusion causes large infarcts due to deficient collaterals, exhibited more profound CSD-induced oligemia and were more vulnerable compared to Swiss mice such that a single CSD was sufficient to induce ischemic lesions at the tip of PAs. In conclusion, CSD-induced prolonged oligemia has potential to cause ischemic/hypoxic injury at hemodynamically vulnerable brain areas, which may be one of the mechanisms underlying WMLs located at the tip of medullary arteries seen in MA patients.

KCl-induced cortical spreading depression waves more heterogeneously propagate than optogenetically-induced waves in lissencephalic brain: an analysis with optical flow tools.

Although cortical spreading depolarizations (CSD) were originally assumed to be homogeneously and concentrically propagating waves, evidence obtained first in gyrencephalic brains and later in lissencephalic brains suggested a rather non-uniform propagation, shaped heterogeneously by factors like cortical region differences, vascular anatomy, wave recurrences and refractory periods. Understanding this heterogeneity is important to better evaluate the experimental models on the mechanistics of CSD and to make appropriate clinical estimations on neurological disorders like migraine, stroke, and traumatic brain injury. This study demonstrates the application of optical flow analysis tools for systematic and objective evaluation of spatiotemporal CSD propagation patterns in anesthetized mice and compares the propagation profile in different CSD induction models. Our findings confirm the asymmetric angular CSD propagation in lissencephalic brains and suggest a strong dependency on induction-method, such that continuous potassium chloride application leads to significantly higher angular propagation variability compared to optogenetically-induced CSDs.

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

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.

Data of ascending cortical vein occlusion induced spreading depression.

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%).

Stress modulates cortical excitability via α-2 adrenergic and glucocorticoid receptors: As assessed by spreading depression.

An increase in cortical excitability may be one of the factors mediating stress-induced vulnerability to neuropsychiatric disorders. Since stress increases extracellular glutamate and predisposes to migraine with aura attacks, we aimed to study the effect of stress on cortical spreading depression (CSD), the biological substrate of migraine aura and a measure of cortical excitability. CSD was induced by increasing concentrations of KCl applied over the dura with 5-minute intervals and recorded from parieto-occipital cortex to assess the CSD-induction threshold in acutely-stressed, chronically-stressed and naive mice. To study the mechanisms of acute stress-induced decrease in CSD threshold, we systemically administered clonidine, yohimbine, propranolol, CRH1 receptor antagonist NBI27914, mifepristone and spironolactone at doses shown to be effective on stress as well as a central noradrenergic neurotoxin (DSP-4) before acute stress. CSD threshold was significantly lowered by acute and chronic stress as well as central noradrenergic denervation. Clonidine and mifepristone further decreased the CSD threshold below the acute stress-induced levels, whereas yohimbine reversed the acute stress-induced decrease in CSD threshold compared to the saline-injected and stressed control groups. Propranolol, NBI27914 and spironolactone did not modify the effect of acute stress on CSD threshold. Stress increases cortical excitability as illustrated by a decrease in CSD-induction threshold. This action of acute stress is mediated by α2-adrenergic and glucocorticoid receptors. The decrease in CSD threshold may account for the stress-induced susceptibility to migraine. CSD may be used as a tool to study the link between stress-related disorders and cortical excitability.

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

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 FeCl3 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.

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

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.

Murine Sialidase Neu3 facilitates GM2 degradation and bypass in mouse model of Tay-Sachs disease.

Tay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal ß-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa-/- mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by ß-hexosaminidase B. Therefore, the presence of the sialidase (s) allows the consequences of the Hexa defect to be bypassed. To determine if the sialidase NEU3 contributes to GM2 ganglioside degradation, we generated a mouse model with combined deficiencies of HEXA and NEU3. The Hexa-/-Neu3-/- mice were healthy at birth, but died at 1.5 to 4.5months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa-/-Neu3-/- mice revealed the abnormal accumulation of GM2 ganglioside. Histological and immunohistochemical analysis demonstrated cytoplasmic vacuolation in the neurons. Electron microscopic examination of the brain, kidneys and testes revealed pleomorphic inclusions of many small vesicles and complex lamellar structures. The Hexa-/-Neu3-/- mice exhibited progressive neurodegeneration with neuronal loss, Purkinje cell depletion, and astrogliosis. Slow movement, ataxia, and tremors were the prominent neurological abnormalities observed in these mice. Furthermore, radiographs revealed abnormalities in the skeletal bones of the Hexa-/-Neu3-/- mice. Thus, the Hexa-/-Neu3-/- mice mimic the neuropathological and clinical abnormalities of the classical early-onset Tay-Sachs patients, and provide a suitable model for the future pre-clinical testing of potential treatments for this condition.

Novel plasminogen gene mutations in Turkish patients with type I plasminogen deficiency.

The plasminogen (Plg) protein is the inactive proenzyme form of plasmin that dissolves fibrin thrombi by a process called fibrinolysis. It has been shown that homozygous or compound-heterozygous deficiency of this protein is a major cause of a rare inflammatory disease affecting mainly mucous membranes found in different body sites. In this study, five individual Turkish patients and nine Turkish families with type 1 Plg deficiency were investigated for PLG gene mutations. All of the coding regions of the PLG gene mutations were screened for mutations using denaturing high-pressure liquid chromatography (DHPLC). Samples showing a different DHPLC profile were subjected to DNA sequencing analysis. Here, we described five novel mutations namely, Cys49Ter, +1 IVS6 G>A, Gly218Val, Tyr283Cys, and Gly703Asp. Previously identified five nonsynonymous (Lys38Glu, Glu180Lys, Gly420Asp, Asp453Asn, Pro763Ser), five synonymous (330 C>T, 582 C>T, 771 T>C, 1083 A>G, 2286 T>G), and a 3' untranslated region (3' UTR) mutation (c.*45 A>G) were also reported in this present study. In this study, we have identified a total of eight mutations, five of which are novel. The mutations/polymorphisms identified in eight of the patients do not explain the disease phenotype. These cases probably carry other pathological mutations (homozygous or compound heterozygous) that cannot be detected by DHPLC.

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

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.