Mannose binding lectin deficiency attenuates neurobehavioral deficits following experimental traumatic brain injury

Introduction: Mannose binding lectin (MBL) is the activator of the lectin complement pathway. After cerebral ischemia it has been shown that MBL could be a mediator of secondary brain damage, in contrast after traumatic brain injury (TBI) there are data suggesting that it could be linked to neuroprotection. We tested the hypothesis that MBL is involved in the pathophysiology of TBI. We 1) characterized the temporal activation of MBL and 2) the effects of its inhibition in a model of experimental TBI. Methods: 1) Male C57/Bl6 mice were subjected to intraperitoneal anesthesia (Pentobarbital, 65 mg/kg) followed by the controlled cortical impact brain injury model of experimental TBI (injury parameters: velocity of 5 meter/second and 1 mm depth of deformation). MBL immunostaining was evaluated at various time points after TBI: 30 minutes, 1, 6, 12, 24, 48, 96 hours and 1 week using anti MBL-A and MBL-C antibodies (n=3). 2) The effects of MBL inhibition were evaluated by comparing functional and histologic outcomes in C57/Bl6 mice (WT) and in MBL knock-out (-/-) mice. Functional outcome was tested using the Composite Neuroscore and Beam Walk test weekly up to 4 weeks postinjury (n = 11). Histologic outcome was evaluated by calculating the contusion volume at 4 weeks postinjury (n = 6). Sham-operated mice received identical anesthesia without brain injury. Results: We observed a robust MBL positive immunostaining in the injured cerebral cortex starting at 30 minutes postinjury and up to 1 week, suggestive of an activation of this pathway following TBI. MBL was observed both at endothelial and tissue level. Consistently, injured WT and MBL (-/-) mice showed neurological motor deficits up to 4 weeks postinjury when compared to their sham controls. Notably, MBL (-/-) mice showed attenuated behavioral deficits when compared to their WT counterpart at 2-4 weeks postinjury (p < 0.01 for both Neuroscore and Beam Walk test). In contrast we observed similar contusion volumes at 4 weeks postinjury (WT = 15.6 \ub1 3.2 cm3 and MBL KO = 13.9 \ub1 3.2 cm3, p = 0.3). Conclusions: We observed that 1) MBL deposition and/or synthesis is increased following TBI; 2) MBL deficiency is associated with functional neuroprotection, suggesting that MBL modulation might be a potential therapeutic target after TBI

Neurosphere-Derived Cells Exert a Neuroprotective Action by Changing the Ischemic Microenvironment

BACKGROUND: Neurosphere-derived cells (NC), containing neural stem cells, various progenitors and more differentiated cells, were obtained from newborn C57/BL6 mice and infused in a murine model of focal ischemia with reperfusion to investigate if: 1) they decreased ischemic injury and restored brain function; 2) they induced changes in the environment in which they are infused; 3) changes in brain environment consequent to transient ischemia were relevant for NC action. METHODOLOGY/PRINCIPAL FINDINGS: NC were infused intracerebroventricularly 4 h or 7 d after 30 min middle cerebral artery occlusion. In ischemic mice receiving cells at 4 h, impairment of open field performance was significantly improved and neuronal loss significantly reduced 7–14 d after ischemia compared to controls and to ischemic mice receiving cells at 7 d. Infusion of murine foetal fibroblast in the same experimental conditions was not effective. Assessment of infused cell distribution revealed that they migrated from the ventricle to the parenchyma, progressively decreased in number but they were observable up to 14 d. In mice receiving NC at 7 d and in sham-operated mice, few cells could be observed only at 24 h, indicating that the survival of these cells in brain tissue relates to the ischemic environment. The mRNA expression of trophic factors such as Insulin Growth Factor-1, Vascular Endothelial Growth Factor-A, Transforming Growth Factor-β1, Brain Derived Neurotrophic Factor and Stromal Derived Factor−1α, as well as microglia/macrophage activation, increased 24 h after NC infusion in ischemic mice treated at 4 h compared to sham-operated and to mice receiving cells at 7 d. CONCLUSIONS/SIGNIFICANCE: NC reduce functional impairment and neuronal damage after ischemia/reperfusion injury. Several lines of evidence indicate that the reciprocal interaction between NC and the ischemic environment is crucial for NC protective actions. Based on these results we propose that a bystander control of the ischemic environment may be the mechanism used by NC to rapidly restore acutely injured brain function

Practical and clinical utility of non-invasive vagus nerve stimulation (nVNS) for the acute treatment of migraine. A post hoc analysis of the randomized, sham-controlled, double-blind PRESTO trial

Background: The PRESTO study of non-invasive vagus nerve stimulation (nVNS; gammaCore®) featured key primary and secondary end points recommended by the International Headache Society to provide Class I evidence that for patients with an episodic migraine, nVNS significantly increases the probability of having mild pain or being pain-free 2 h post stimulation. Here, we examined additional data from PRESTO to provide further insights into the practical utility of nVNS by evaluating its ability to consistently deliver clinically meaningful improvements in pain intensity while reducing the need for rescue medication. Methods: Patients recorded pain intensity for treated migraine attacks on a 4-point scale. Data were examined to compare nVNS and sham with regard to the percentage of patients who benefited by at least 1 point in pain intensity. We also assessed the percentage of attacks that required rescue medication and pain-free rates stratified by pain intensity at treatment initiation. Results: A significantly higher percentage of patients who used acute nVNS treatment (n = 120) vs sham (n = 123) reported a ≥ 1-point decrease in pain intensity at 30 min (nVNS, 32.2%; sham, 18.5%; P = 0.020), 60 min (nVNS, 38.8%; sham, 24.0%; P = 0.017), and 120 min (nVNS, 46.8%; sham, 26.2%; P = 0.002) after the first attack. Similar significant results were seen when assessing the benefit in all attacks. The proportion of patients who did not require rescue medication was significantly higher with nVNS than with sham for the first attack (nVNS, 59.3%; sham, 41.9%; P = 0.013) and all attacks (nVNS, 52.3%; sham, 37.3%; P = 0.008). When initial pain intensity was mild, the percentage of patients with no pain after treatment was significantly higher with nVNS than with sham at 60 min (all attacks: nVNS, 37.0%; sham, 21.2%; P = 0.025) and 120 min (first attack: nVNS, 50.0%; sham, 25.0%; P = 0.018; all attacks: nVNS, 46.7%; sham, 30.1%; P = 0.037). Conclusions: This post hoc analysis demonstrated that acute nVNS treatment quickly and consistently reduced pain intensity while decreasing rescue medication use. These clinical benefits provide guidance in the optimal use of nVNS in everyday practice, which can potentially reduce use of acute pharmacologic medications and their associated adverse events. Trial registration: ClinicalTrials.gov identifier: NCT02686034

A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

BACKGROUND: Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection

Soluble CD59 Expressed from an Adenovirus In Vivo Is a Potent Inhibitor of Complement Deposition on Murine Liver Vascular Endothelium

Inappropriate activation of complement on the vascular endothelium of specific organs, or systemically, underlies the etiology of a number of diseases. These disorders include atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, atherosclerosis, age-related macular degeneration, diabetic retinopathy, and transplant rejection. Inhibition of the terminal step of complement activation, i.e. formation of the membrane attack complex, using CD59 has the advantage of retaining the upstream processes of the complement cascade necessary for fighting pathogens and retaining complement's crucial role in tissue homeostasis. Previous studies have shown the necessity of membrane targeting of soluble CD59 in order for it to prove an effective inhibitor of complement deposition both in vitro and in vivo. In this study we have generated an in vivo model of human complement activation on murine liver vascular endothelium. This model should prove useful for the development of anti-complement therapies for complement-induced pathologies of vascular endothelium. Using this model, we have demonstrated the viability of a non membrane-targeted soluble CD59 to significantly inhibit complement deposition on the endothelium of murine liver vasculature when expressed in vivo from an adenovirus. This result, unanticipated based on prior studies, suggests that the use of non membrane-targeted sCD59 as an anti-complement therapy be re-visited

Lovastatin Modulates Glycogen Synthase Kinase-3β Pathway and Inhibits Mossy Fiber Sprouting after Pilocarpine-Induced Status Epilepticus

This study was undertaken to assay the effect of lovastatin on the glycogen synthase kinase-3 beta (GSK-3β) and collapsin responsive mediator protein-2 (CRMP-2) signaling pathway and mossy fiber sprouting (MFS) in epileptic rats. MFS in the dentate gyrus (DG) is an important feature of temporal lobe epilepsy (TLE) and is highly related to the severity and the frequency of spontaneous recurrent seizures. However, the molecular mechanism of MFS is mostly unknown. GSK-3β and CRMP-2 are the genes responsible for axonal growth and neuronal polarity in the hippocampus, therefore this pathway is a potential target to investigate MFS. Pilocarpine-induced status epilepticus animal model was taken as our researching material. Western blot, histological and electrophysiological techniques were used as the studying tools. The results showed that the expression level of GSK-3β and CRMP-2 were elevated after seizure induction, and the administration of lovastatin reversed this effect and significantly reduced the extent of MFS in both DG and CA3 region in the hippocampus. The alteration of expression level of GSK-3β and CRMP-2 after seizure induction proposes that GSK-3β and CRMP-2 are crucial for MFS and epiletogenesis. The fact that lovastatin reversed the expression level of GSK-3β and CRMP-2 indicated that GSK-3β and CRMP-2 are possible to be a novel mechanism of lovatstain to suppress MFS and revealed a new therapeutic target and researching direction for studying the mechanism of MFS and epileptogenesis

Tumor necrosis factor-α-mediated threonine 435 phosphorylation of p65 nuclear factor-κB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus

<p>Abstract</p> <p>Background</p> <p>Status epilepticus (SE) induces severe vasogenic edema in the piriform cortex (PC) accompanied by neuronal and astroglial damages. To elucidate the mechanism of SE-induced vasogenic edema, we investigated the roles of tumor necrosis factor (TNF)-α in blood-brain barrier (BBB) disruption during vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced SE.</p> <p>Methods</p> <p>SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, and soluble TNF p55 receptor (sTNFp55R) prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunits.</p> <p>Results</p> <p>Following SE, most activated microglia showed strong TNF-α immunoreactivity. In addition, TNF p75 receptor expression was detected in endothelial cells as well as astrocytes. In addition, only p65-Thr435 phosphorylation was increased in endothelial cells accompanied by SMI-71 expression (an endothelial barrier antigen). Neutralization of TNF-α by soluble TNF p55 receptor (sTNFp55R) infusion attenuated SE-induced vasogenic edema and neuronal damages via inhibition of p65-Thr435 phosphorylation in endothelial cells. Furthermore, sTNFp55R infusion reduced SE-induced neutrophil infiltration in the PC.</p> <p>Conclusion</p> <p>These findings suggest that impairments of endothelial cell functions via TNF-α-mediated p65-Thr 485 NF-κB phosphorylation may be involved in SE-induced vasogenic edema. Subsequently, vasogenic edema results in extensive neutrophil infiltration and neuronal-astroglial loss.</p

Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 beta

<p>Abstract</p> <p>Objective</p> <p>Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K⁺ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β) on Kir4.1 mRNA and protein expression.</p> <p>Methods</p> <p>We investigated Kir4.1 (Kcnj10) and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (<it>n</it> = 64), comparing the expression in tumor patients with (<it>n</it> = 38) and without epilepsy (<it>n</it> = 26).</p> <p>Results</p> <p>Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1).</p> <p>Conclusions</p> <p>Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the inflammatory cytokine IL-1β.</p