Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end.

Microtubule polymerization dynamics result from the biochemical interactions of αß-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αß-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP-tubulin at the growing microtubule end may be an important early event determining catastrophe.

Introducing a Realistic, Low-Cost Simulation Model for Clipping of Brain Aneurysms.

BACKGROUND: The current trend toward endovascular treatment of brain aneurysms may have a negative impact on young neurosurgeons who are less exposed to these lesions, thus affecting the acquisition of surgical skills in the field. Different training models have emerged to help cope with this issue, but these have specific pitfalls. Training models based on live animals or cadaveric specimens face increasing restrictions as regulations become a barrier in accessibility for everyday skills development. We introduce a novel, realistic, and inexpensive simulation model using a fresh bovine brain, and we assess its face and content validity as a training tool. METHODS: A fresh bovine brain is used to simulate microsurgical fissure dissection. Arterial and aneurysmal components are created with arteries and veins harvested from chicken thigh. A 12-item questionnaire using the Likert numeric scale (grades 1 - 5) was used to assess the validity of model in 10 surgeons. RESULTS: Ten neurosurgeons performed the simulated clipping of the aneurysm and completed a questionnaire. All surgeons surveyed responded "agree" or "strongly agree" that the simulator, and the skills trained with it, are comparable to clipping brain aneurysms. All respondents believed that this simulator could improve patient safety. CONCLUSIONS: We present a novel, realistic, and inexpensive simulation model for the clipping of brain aneurysms. This model was partially validated by the opinion of field experts. We believe this model has the potential to become a useful training tool for young neurosurgeons who have little exposure to real aneurysm cases.

Modelo de simulación de clipado de aneurismas cerebrales de bajo costo, validez aparente y de contenido / 1st Prize Scholarship: Low-cost brain aneurysm clipping simulation model, face and content validity

Objetivo: Presentar un modelo de entrenamiento realista en clipado microquirúrgico de aneurisma cerebral con flujo pulsátil, artificial, accesible y de bajo costo. Evaluar su validez aparente y de contenido. Introducción: La tendencia actual hacia el tratamiento endovascular de aneurismas cerebrales puede reducir la exposición de neurocirujanos jóvenes al tratamiento quirúrgico de estas lesiones durante su formación, afectando la adquisición de habilidades quirúrgicas en este campo. Presentamos un modelo de simulación fácilmente reproducible para clipado de aneurismas cerebrales verificando su validez como herramienta de entrenamiento. Material y métodos: Se utilizó cerebro bovino como alternativa al cerebro humano para simular apertura de surcos. Para la confección de aneurismas se utilizaron arteria y vena femoral de muslo de pollo. Como sistema de micro flujo se utilizó una mini bomba sumergible. Discusión: La simulación con animales vivos se considera entre los modelos más realistas para el entrenamiento en microcirugía, pero las restricciones y su costo limitan su uso, tal como ocurre con los especímenes cadavéricos humanos. Con el propósito de resolver estas dificultades, desarrollamos un modelo de bajo costo y lo usamos para similar el clipado de aneurismas cerebrales. Diez neurocirujanos experimentados utilizaron el mismo y luego completaron una encuesta para evaluar su potencial beneficio. Conclusión: Hemos presentado un nuevo modelo de simulación para el clipado microquirúrgico de aneurismas cerebrales utilizando materiales accesibles y económicos. Creemos que el mismo resulta útil y fácilmente reproducible para la práctica y el aprendizaje de la técnica microquirúrgica para neurocirujanos en formación

Objective: To show a realistic and accessible training model for cerebral aneurysm clipping with a pulsatile flow. To determine its face and content validity. Background: The current trend towards endovascular treatment of brain aneurysms may have a negative impact on young neurosurgeons who are less exposed to these lesions, thus affecting the acquisition of surgical skills in the field. We introduce an easily reproducible simulation model for clipping of cerebral aneurysms and we assess its validity as a training tool. Material and methods: A fresh bovine brain is used to simulate microsurgical fissure dissection. Arterial and aneurysmal components are created with arteries and veins harvested from chicken thigh. For the micro flow system, a submersible mini-pump was employed. Discussion: Live animal simulations are considered among the most realistic training models but restrictions and the facilities costs limit their use, just as with human cadaveric specimens. With the aim of addressing these issues, we developed a low-cost model with the use of a micro flow pump and used it for simulation of aneurysm clipping. Ten neurosurgeons performed the simulated clipping of the aneurysm and were administered a questionnaire following the procedure. Conclusion: We present a novel, realistic, inexpensive and easily reproducible simulation model for the clipping of brain aneurysms. This model was partially validated by the opinion of field experts. We believe this model has the potential of becoming a useful training tool for young neurosurgeons

Dynamic mechanical characterization and viscoelastic modeling of bovine brain tissue.

Brain tissue is vulnerable and sensitive, predisposed to potential damage under various conditions of mechanical loading. Although its material properties have been investigated extensively, the frequency-dependent viscoelastic characterization is currently limited. Computational models can provide a non-invasive method by which to analyze brain injuries and predict the mechanical response of the tissue. The brain injuries are expected to be induced by dynamic loading, mostly in compression and measurement of dynamic viscoelastic properties are essential to improve the accuracy and variety of finite element simulations on brain tissue. Thus, the aim of this study was to investigate the compressive frequency-dependent properties of brain tissue and present a mathematical model in the frequency domain to capture the tissue behavior based on experimental results. Bovine brain specimens, obtained from four locations of corona radiata, corpus callosum, basal ganglia and cortex, were tested under compression using dynamic mechanical analysis over a range of frequencies between 0.5 and 35 Hz to characterize the regional and directional response of the tissue. The compressive dynamic properties of bovine brain tissue were heterogenous for regions but not sensitive to orientation showing frequency dependent statistical results, with viscoelastic properties increasing with frequency. The mean storage and loss modulus were found to be 12.41 kPa and 5.54 kPa, respectively. The material parameters were obtained using the linear viscoelastic model in the frequency domain and the numeric simulation can capture the compressive mechanical behavior of bovine brain tissue across a range of frequencies. The frequency-dependent viscoelastic characterization of brain tissue will improve the fidelity of the computational models of the head and provide essential information to the prediction and analysis of brain injuries in clinical treatments.

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides.

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series. Graphical abstract.

A Simple Adaptable Blood-Brain Barrier Cell Model for Screening Matrix Metalloproteinase Inhibitor Functionality.

The blood-brain barrier is a multicellular and basement membrane unit that regulates molecular transport between the blood and central nervous system. Many cerebral pathologies, such as acute stroke and chronic vascular dementia, result in a disrupted blood-brain barrier, increasing its permeability and allowing the entry of potentially neurotoxic molecules. The activation of matrix metalloproteinases mediates further blood-brain barrier damage. The inhibition of matrix metalloproteinases is a potential strategy for stroke therapy. As inhibitors are developed, efficient context-specific screening methods will be required. Models of the blood-brain barrier have been extensively used to study neuropathologies and the effect of various treatment options.Herein, we describe a co-culture model of the blood-brain barrier composed of brain microvascular endothelial cells and astrocytes grown on an artificial basement membrane-coated membrane insert. Our cell model forms a barrier and is a simple first approximation of blood-brain barrier integrity. As currently developed, the model may be applied to testing the effect of matrix metalloproteinases and matrix metalloproteinase inhibitors on blood-brain barrier physiology and pathophysiology. The model is a quick and effective evaluation tool for generating nonclinical data in a living cell system before proceeding to animal models.

Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers.

Receptor-mediated transcytosis of the transferrin receptor has been suggested as a potential transport system to deliver therapeutic molecules into the brain. Recent studies have however shown that therapeutic antibodies, which have been reported to cross the brain endothelium, reach greater brain exposure when the affinity of the antibodies to the transferrin receptor is lowered. The lower affinity of the antibodies to the transferrin receptor facilitates the dissociation from the receptor within the endosomal compartments, which may indicate that the receptor itself does not necessarily move across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin receptor localization patterns in mono-cultures and co-cultures. The endothelial cells demonstrated an up-regulation of transferrin receptor mRNA after treatment with the iron chelator deferoxamine. The association of [125I]holo-transferrin and [59Fe]-transferrin to the endothelial cells was inhibited by an excess of unlabeled holo-transferrin, indicating receptor mediated association. However, over time the cell associated [59Fe]-label exceeded that of [125I]holo-transferrin, which could indicate release of iron in the endothelial cells and receptor recycling. Luminal-to-abluminal transport of [125I]holo-transferrin across endothelial cell monolayers was low and not inhibited by unlabeled holo-transferrin. This indicated that transendothelial transferrin transport was independent of transferrin receptor-mediated transcytosis.

Fucoidan Extracted from Hijiki Protects Brain Microvessel Endothelial Cells Against Diesel Exhaust Particle Exposure-Induced Disruption.

This study was performed to evaluate the protective effects of fucoidan against the decreased function of primary cultured bovine brain microvessel endothelial cells (BBMECs) after exposure to diesel exhaust particles (DEPs). BBMECs were extracted from bovine brains and cultured until confluent. To evaluate the function of BBMECs, we performed a permeability test using cell-by-cell equipment and by Western blot analysis for zonular occludens-1 (ZO-1), which is a tight junction protein of BMECs, and evaluated oxidative stress in BBMECs using the DCFH-DA assay and the CUPRAC-BCS assay. The increased oxidative stress in BBMECs following DEP exposure was suppressed by fucoidan. In addition, permeability of BBMECs induced by DEP exposure was decreased by fucoidan treatment. Our results showed that fucoidan protects against BBMEC disruption induced by DEP exposure. This study provides evidence that fucoidan might protect the central nervous system (CNS) against DEP exposure.

Kinesin-1 translocation: Surprising differences between bovine brain and MCF7-derived microtubules.

While there have been many single-molecule studies of kinesin-1, most have been done along microtubules purified from bovine or porcine brain, and relatively little is known about how variations in tubulin might alter motor function. Of particular interest is transport along microtubules polymerized from tubulin purified from MCF7 breast cancer cells, both because these cells are a heavily studied model system to help understand breast cancer, and also because the microtubules are already established to have interesting polymerization/stability differences from bovine tubulin, suggesting that perhaps transport along them is also different. Thus, we carried out paired experiments to allow direct comparison of in vitro kinesin-1 translocation along microtubules polymerized from either human breast cancer cells (MCF7) or microtubules from bovine brain. We found surprising differences: on MCF7 microtubules, kinesin-1's processivity is significantly reduced, although its velocity is only slightly altered.

Endothelial calcium dynamics, connexin channels and blood-brain barrier function.

Situated between the circulation and the brain, the blood-brain barrier (BBB) protects the brain from circulating toxins while securing a specialized environment for neuro-glial signaling. BBB capillary endothelial cells exhibit low transcytotic activity and a tight, junctional network that, aided by the cytoskeleton, restricts paracellular permeability. The latter is subject of extensive research as it relates to neuropathology, edema and inflammation. A key determinant in regulating paracellular permeability is the endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)]i) that affects junctional and cytoskeletal proteins. Ca(2+) signals are not one-time events restricted to a single cell but often appear as oscillatory [Ca(2+)]i changes that may propagate between cells as intercellular Ca(2+) waves. The effect of Ca(2+) oscillations/waves on BBB function is largely unknown and we here review current evidence on how [Ca(2+)]i dynamics influence BBB permeability.

Experimental study of the compositive applying of nourishing Piyin Remedy and bovine brain extract on spinal cord injury of rats / 中国康复理论与实践

@# ObjectiveTo study the effect and mechanisms of nourishing Piyin Remedy (nPR) and bovine brain extract (bBE) on experimental spinal cord injury (SCI) of rat.Methods80 healthy SD rats were divided into 5 equal groups randomly: bBE group supplied through subarachnoid cavity, normal saline (NS) group supplied through subarachnoid cavity, nPR group, NS orally taken group, combined group. Animal models were made by Allen's equipment on T8～T9 segment. The spinal nerve function, somatosensory evoked potentials (SEP), retrograde and label technique of horseradish peroxidase, gross observation, histological and morphometric analysis were taken as the observed indices.ResultsThe values of observed indices of bBE group and nPR group improved evidently compared with their own control groups; that of combined group was prior to sole administration.ConclusionnPR can hold back the secondary SCI and accelerate the recovery of spinal nerve function; bBE can stimulate the improvement of injuried nerve fibers; the joint of nPR and bBE can make a synergic effect.

Experimental Immune-Mediated Hearing Loss / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: In 1979, MaCabe described the autoimmune sensorineural hearing loss generally characterized by bilateral progressive sensorineural hearing loss over weeks to months. He also described steroid responsiveness of heaing loss. Since then, numerous attempts have been tried to investigate autoimmune inner ear disease. But, there is insufficient information concerning pathophysiology and no reliable laboratory tests are available for diagnosis. In this study, we immunized healthy rats with bovine brain antigen, and monitored auditory brainstem response threshold shifts and serum antibody titer especially antibody to 68 kD protein, and observed histologic changes to develop animal model and to investigate pathophysiology of inner ear autoimmunity. MATERIALS & METHODS:We used 15 female Wistar rats weighing 200 g to 250 g. Three of them (as control group) were immunized with bovine serum albumin and the rest were weekly immunized with bovine brain antigen 3 times. After the antigen challenges, animals were sacrificed at 1st, 2nd, 4th or 8th week after collection of serum and ABR test. Hearing was evaluated by ABR prior to each immunization and at 1st, 2nd, 3rd, 4th, 6th or 8th week following immunization. Collected sera were analyzed by Western blotting immunoassay against fresh bovine brain antigen preparation. RESULTS: On ABR, hearing threshold shifts of 10 to 40 dB were recorded and shifts of greater than 20 dB were recorded in 8 ears (33.3%) of 24 ears. On Western blot assay, a band at 68 kD M.W. was observed with high binding activity at 1st week through 4th week following immunization. Cellular infiltration was observed in the are as adjacent to the spiral modiolar venules in the cochlear modiolus and in the spiral ganglion cells in a scattered pattern. CONCLUSION: These results suggest that bovine brain antigen can induce autoimmune inner ear disease in experimental animals and that inner ear autoimmunity may play an important role in the development of inner ear disorders and hearing loss.