Comparative in vitro studies on PBN loaded nanoparticles prepared by biodegradable chitosan, PLGA polymers and their PEGylated block copolymers

α-phenyl-N-tert-butyl nitrone (PBN) is a neuroprotective free radical scavenger however it has low in vivo stability and blood residence time. Aim. of this study is to develop a nanoparticle formulation by using different polymeric system which enhance the blood residence time and in vivo stability of PBN and characterize in terms of particle size, zeta potential, morphology, encapsulation efficiency, in vitro release profiles. Chitosan (CS), poly(D,L-lactide-co-glycolide) (PLGA) and their poly(ethylene glycol) (PEG) block co-polymers were used for comparative study. Results showed that particle sizes of CS, CS-PEG, PLGA and PLGA-PEG nanoparticles are between 142-356 nm. PLGA nanoparticles and their block copolymers' nanoparticle have greatly monodisperse distribution. CS and CS-PEG nanoparticles have zeta potential values between 17-40 mV related to amine groups, contrariwise PLGA and PLGA-PEG nanoparticles have negative zeta potential in the range of (-8)-(-19) mV. Encapsulation efficiency and loading capacity for all formulations are between 12-54 %, 9-68 %, respectively. PLGA-PEG nanoparticles are promising for further studies due to their sufficient encapsulation efficiency and in vitro release profilesAuthors would like to acknowledge that this project was financially supported by Tubitak (Scientific Research Project Number: 110S460)S

An aquaporin 4 antisense oligonucleotide loaded, brain targeted nanoparticulate system design

Aquaporins (AQPs), members of the water-channel protein family, are highly expressed in brain tissue especially in astrocytic end-feet. They are important players for water hemostasis during development of cytotoxic as well as vasogenic edema. Increased expression of AQPs is important in pathophysiology of neurological diseases such as neuroinflammation and ischemia. Unfortunately, there are a few pharmacological inhibitors of AQP4 with several side effects limiting their translation as a drug for use in clinical conditions. Another therapeutic approach is using antisense oligonucleotides (ASOs) to block AQP4 activity. These are short, synthetic, modified nucleic acids that bind RNA to modulate its function. However, they cannot pass the blood brain barrier (BBB). To overcome this obstacle we designed a nanoparticulate system made up of chitosan nanoparticles surface modified with PEG and conjugated with monoclonal anti transferrin receptor-1 antibody via streptavidin-biotin binding. The nanocarrier system could be targeted to the transferrin receptor-1 at the brain endothelial capillaries through monoclonal antibodies. It is hypothesized that the nanoparticles could pass the BBB via receptor mediated transcytosis and reach brain parenchyma. Particle size, zeta potential, loading capacity and release profiles of nanoparticles were investigated. It was observed that all types of chitosau (CS) nanoparticles had positive zeta potential values and nanoparticle particle size distribution varied between 100 and 800 nm. The association efficiency of ASOs into the nanoparticles was between 80–97% and the release profiles of the nanoparticles exhibited an initial burst effect followed by a controlled release. The results showed that the designed chitosan based nanocarriers could be a promising carrier system to transport nucleic acid based drugs to brain parenchymaThis study is supported by The Scientific and Technological Research Council of Turkey (TUBITAK, Project Number: 110S460)S

Systemically Administered Brain-Targeted Nanoparticles Transport Peptides across the Blood—Brain Barrier and Provide Neuroprotection

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 strokeTurgay Dalkara’s work is supported by the Turkish Academy of Sciences. This study is supported by The Scientific and Technological Research Council of Turkey (TUBITAK, Project Number: 109S017)S

The Presence Of Autoantibodies Against Vascular And Nervous Tissue In Sera From Patients With Neuro-Behcet'S Disease

Introduction: Behcet's disease is a chronic inflammatory disease of unknown aetiology that affects multiple organ systems. Since the diagnosis of this disease mainly relies on clinical criteria, a diagnostic laboratory test is required especially for neuro-Behcet's patients without systemic involvement. Method: In this study, we searched for the presence of autoantibodies against brain tissue, by means of indirect immunofluorescent staining technique in sera obtained from patients with neuro-Behcet's disease, based on reports that humoral immune dysregulation may play a role in susceptibility to Behcet's disease. After pre-absorbtion of sera with guinea pig liver powder to reduce nonspecific staining, serum samples were applied to mouse brain sections and immunoreactivity was detected with fluorescein (FITC)-conjugated goat antibody against human IgG. Results: Ten sera from neuro-Behcet's patients and 10 age-matched control sera were screened for immunoreactivity. We detected specific immunoreactivity to both parenchymal and vascular brain structures in the patients' sera. Parenchymal vessel immunopositivity was detected in 8 of 10 patients, whereas only two of control sera showed no significant parenchymal vascular immunoreactivity (p=0.025). In addition to vascular immunoreactivity, filamentous and reticular immunopositive structures were detected in brain sections of 5 out of 10 patients. No such immunoreactivity was detected in sections incubated with control sera (p=0.016). Conclusion: We detected a specific immunoreactivity against vascular and parenchymal filamentous structures in neuro-Behcet patients' sera. Humoral autoimmunity may play a role in the pathogenesis of neuro-Behcet's disease in addition to cellular immune response. Findings of this preliminary study will be evaluated with a large number of patients and controls, to determine whether it is the cause or the result and, further studies are underway to disclose the nature of epitope to which the immunoreactivity was directed against and to develop a diagnostic laboratory method for investigating central nervous system involvement in Behcet's patients.Wo

Optical Coherence Tomography Imaging Of Capillary Reperfusion After Ischemic Stroke

Although progress has been made for recanalization therapies after ischemic stroke, post-treatment imaging studies show that tissue reperfusion cannot be attained despite satisfactory recanalization in a significant percentage of patients. Hence, investigation of microcirculatory changes in both surface and deep cortical levels after ischemia reperfusion is important for understanding the post-stroke blood flow dynamics. In this study, we applied optical coherence tomography (OCT) imaging of cerebral blood flow for the quantification of the microcirculatory changes. We obtained OCT microangiogram of the brain cortex in a mouse stroke model and analyzed the data to trace changes in the capillary perfusion level (CPL) before, during, and after the stroke. The CPL changes were estimated in 1 and 2 h ischemia groups as well as in a non-ischemic sham-operated group. For the estimation of CPL, a decorrelation amplitude-based algorithm was implemented and used. As a result, the CPL considerably decreased during ischemia but recovered to the baseline when recanalization was performed 1 h after ischemia; however, the CPL was significantly reduced when recanalization was delayed to 2 h after ischemia. These data demonstrate that ischemia causes microcirculation dysfunction, leading to a decreased capillary reperfusion after recanalization. Microcirculatory no-reflow warrants more rigorous assessment in clinical trials, whereas advanced optical imaging techniques may provide mechanistic insight and solutions in experimental studies. (C) 2016 Optical Society of AmericaWoSScopu

Data Of Indirect Immunofluorescence Labeling Of The Mouse Brain Sections With Sera From Sle And Ms Patients

The data presented in this article are related to the research article entitled “Behcet Disease serum is immunoreactive to neurofilament medium which share common epitopes to bacterial HSP-65, a putative trigger” (Lule et a. 2017) . The immunoreactivity to self-antigens is well characterized for systemic lupus erythematosus (SLE) and multiple sclerosis (MS) (Magro Checa et al., 2013) . Indirect immunofluorescence labeling of the mouse tissue sections with patient sera has recently been popular to discover novel epitopes and gain mechanistic insight to diseases with dysregulated immunity (Lennon et al., 2004) . The present article demonstrates widespread labeling of cell nuclei with SLE patient sera and sporadic filamentous labeling along the axons with MS patient sera on mouse brain sections. The filamentous immunolabeling was sometimes associated with cytoplasmic staining of cells, which sent processes along the axon bundles, suggesting that they were oligodendrocytes. Since the mouse brain tissue has little autofluorescence and limited connective tissue causing non-specific immunolabeling, it appears superior to peripheral tissues for searching serum immunoreactivity.PubMe

The effect of P2X7 antagonism on subcortical spread of optogenetically-triggered cortical spreading depression and neuroinflammation

Abstract Migraine is a neurological disorder characterized by episodes of severe headache. Cortical spreading depression (CSD), the electrophysiological equivalent of migraine aura, results in opening of pannexin 1 megachannels that release ATP and triggers parenchymal neuroinflammatory signaling cascade in the cortex. Migraine symptoms suggesting subcortical dysfunction bring subcortical spread of CSD under the light. Here, we investigated the role of purinergic P2X7 receptors on the subcortical spread of CSD and its consequent neuroinflammation using a potent and selective P2X7R antagonist, JNJ-47965567. P2X7R antagonism had no effect on the CSD threshold and characteristics but increased the latency to hypothalamic voltage deflection following CSD suggesting that ATP acts as a mediator in the subcortical spread. P2X7R antagonism also prevented cortical and subcortical neuronal activation following CSD, revealed by bilateral decrease in c-fos positive neuron count, and halted CSD-induced neuroinflammation revealed by decreased neuronal HMGB1 release and decreased nuclear translocation of NF-kappa B-p65 in astrocytes. In conclusion, our data suggest that P2X7R plays a role in CSD-induced neuroinflammation, subcortical spread of CSD and CSD-induced neuronal activation hence can be a potential target

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