Bovine Spinal Cord Research Articles

Evaluating the angiogenic and mechanical properties of hydrogels and physical constructs derived from spinal cord meninges extracellular matrix

The vasculature is an integral unit of the tissue microenvironment due to providing nutrients and oxygen to surrounding cells. Therefore, pro-angiogenic biomaterials have the potential to improve the success of a wide range of medical therapies, including tissue engineering, wound healing, and drug delivery. Herein, we decellularized bovine spinal cord meninges with Triton X-100 and digested them with pepsin to obtain a hydrogel (MeninGEL). The cryogel form of the MeninGEL was also prepared by lyophilization process (named as MeninRIX). DNA content analysis showed that the nuclear content was significantly reduced by 98.6% after decellularization process. Furthermore, the effect of decellularization on extracellular matrix components was investigated with glycosaminoglycan (GAG) and hydroxyproline (HYP) content analyses. Tensile, compression, and suture retention tests were performed to elucidate the mechanical properties. The physiological degradation behavior of the bioscaffolds was investigated by hydrolytically. Both MeninGEL and MeninRIX have good biocompatibility and pro-angiogenic properties, as proved by the Chick Chorioallantoic Membrane (CAM) assay. Moreover, SEM and histological analyses indicated cellular migration, attachment, and dynamism on the bioscaffolds’ surfaces. On the basis of these data, MeninGEL and MeninRIX are pro-angiogenic structures and have adequate mechanical properties, which makes them promising candidates for soft regenerative medicine applications.

The mechanical behavior of bovine spinal cord white matter under various strain rate conditions: tensile testing and visco-hyperelastic constitutive modeling.

The mechanical behavior of the white matter is important for estimating the damage of the spinal cord during accidents. In this study, we conducted uniaxial tension testing in vitro of bovine spinal cord white matter under extremely high strain rate conditions (up to 100s-1). A visco-hyperelastic constitutive law for modeling the strain rate-dependent behavior of the bovine spinal cord white matter was developed. A set of material constants was obtained using a Levenberg-Marquardt fitting algorithm to match the uniaxial tension experimental data with various strain rates. Our experimental data confirmed that the modulus and tensile strength increased when the strain rate is higher. For the extremely high strain rate condition (100s-1), we found that both the modulus and failure stress significantly increased compared with the low strain rate case. These new data in terms of mechanical response at high strain rate provide insight into the spine injury mechanism caused by high-speed impact. Moreover, the developed constitutive model will allow researchers to perform more realistic finite element modeling and simulation of spinal cord injury damage under various complicated conditions.

Comparison of numerical methods for cerebrospinal fluid representation and fluid-structure interaction during transverse impact of a finite element spinal cord model.

Spinal cord impacts can have devastating consequences. Computational models can investigate such impacts but require biofidelic numerical representations of the neural tissues and fluid-structure interaction with cerebrospinal fluid. Achieving this biofidelity is challenging, particularly for efficient implementation of the cerebrospinal fluid in full computational human body models. The goal of this study was to assess the biofidelity and computational efficiency of fluid-structure interaction methods representing the cerebrospinal fluid interacting with the spinal cord, dura, and pia mater using experimental pellet impact test data from bovine spinal cords. Building on an existing finite element model of the spinal cord and pia mater, an orthotropic hyperelastic constitutive model was proposed for the dura mater and fit to literature data. The dura mater and cerebrospinal fluid were integrated with the existing finite element model to assess four fluid-structure interaction methods under transverse impact: Lagrange, pressurized volume, smoothed particle hydrodynamics, and arbitrary Lagrangian-Eulerian. The Lagrange method resulted in an overly stiff mechanical response, whereas the pressurized volume method over-predicted compression of the neural tissues. Both the smoothed particle hydrodynamics and arbitrary Lagrangian-Eulerian methods were able to effectively model the impact response of the pellet on the dura mater, outflow of the cerebrospinal fluid, and compression of the spinal cord; however, the arbitrary Lagrangian-Eulerian compute time was approximately five times higher than smoothed particle hydrodynamics. Crucial to implementation in human body models, the smoothed particle hydrodynamics method provided a computationally efficient and representative approach to model spinal cord fluid-structure interaction during transverse impact.

Decellularized spinal cord meninges extracellular matrix hydrogel that supports neurogenic differentiation and vascular structure formation.

Decellularization of extracellular matrices offers an alternative source of regenerative biomaterials that preserve biochemical structure and matrix components of native tissues. In this study, decellularized bovine spinal cord meninges (dSCM)-derived extracellular matrix hydrogel (MeninGEL) is fabricated by employing a protocol that involves physical, chemical, and enzymatic processing of spinal meninges tissue and preserves the biochemical structure of meninges. The success of decellularization is characterized by measuring the contents of residual DNA, glycosaminoglycans, and hydroxyproline, while a proteomics analysis is applied to reveal the composition of MeninGEL. Frequency and temperature sweep rheometry show that dSCM forms self-supporting hydrogel at physiological temperature. The MeninGEL possesses excellent cytocompatibility. Moreover, it is evidenced with immuno/histochemistry and gene expression studies that the hydrogel induces growth-factor free differentiation of human mesenchymal stem cells into neural-lineage cells. Furthermore, MeninGEL instructs human umbilical vein endothelial cells to form vascular branching. With its innate bioactivity and low batch-to-batch variation property, the MeninGEL has the potential to be an off-the-shelf product in nerve tissue regeneration and restoration.

Tensile mechanical analysis of anisotropy and velocity dependence of the spinal cord white matter: a biomechanical study.

In spinal cord injuries, external forces from various directions occur at various velocities. Therefore, it is important to physically evaluate whether the spinal cord is susceptible to damage and an increase in internal stress for external forces. We hypothesized that the spinal cord has mechanical features that vary under stress depending on the direction and velocity of injury. However, it is difficult to perform experiment because the spinal cord is very soft. There are no reports on the effects of multiple external forces. In this study, we used bovine spinal cord white matter to test and analyze the anisotropy and velocity dependence of the spinal cord. Tensile-vertical, tensile-parallel, shear-vertical, and shear-parallel tests were performed on the white matter in the fibrous direction (cranial to caudal). Strain rate in the experiment was 0.1, 1, 10, and 100/s. We calculated the Young's modulus of the spinal cord. Results of the tensile and shear tests revealed that stress tended to increase when external forces were applied parallel to the direction of axon fibers, such as in tensile-vertical and shear-vertical tests. However, external forces those tear against the fibrous direction and vertically, such as in tensile-parallel and shear-parallel tests, were less likely to increase stress even with increased velocity. We found that the spinal cord was prone to external forces, especially in the direction of the fibers, and to be under increased stress levels when the velocity of external forces increased. From these results, we confirmed that the spinal cord has velocity dependence and anisotropy. The Institutional Animal Care and Use Committee of Yamaguchi University waived the requirement for ethical approval.

Aging of bovine spinal cord: an alteration of myelinated nerve fibers in the white matter

To gain more knowledge of age-related changes in the spinal cord of the bovine species, the structure of 15 clinically normal aged cattle (Holstein-Friesian cows 8–13 years of age) was histopathologically examined. In 12 (80%) of the 15 animals, the common histopathological features identified were alteration of a small number of myelinated nerve fibers in the white matter, with less than 4 nerve fibers affected per cross-sectional area of the spinal cord. Alteration of nerve fibers was characterized by dilation of myelin sheaths with loss of axons or macrophage infiltration, resembling features of Wallerian axonal degeneration. The occurrence of this nerve fiber alteration had a predilection for the lateral and ventral white matter funiculi. In eight cattle, so-called axonal spheroids were rarely present in the white matter. There was little evidence of glial reaction against nerve fiber alteration. Gray horn neurons were unremarkable. Lipofuscin granules were recognized in neurons, glial cells, and neuropil of the medulla oblongata examined in six cows. The changes observed in the spinal cord white matter of the present cows were similar to those described previously in aged human beings and domestic and laboratory animals, and thus were likely to have been a phenomenon which was closely related to aging. The strict clinical significance of the histopathological changes in the spinal cord white matter remains undetermined.

A novel method for constructing an acellular 3D biomatrix from bovine spinal cord for neural tissue engineering applications.

In this study, we aimed at generating 3-dimensional (3D) decellularized bovine spinal cord extracellular matrix-based scaffolds (3D-dCBS) for neural tissue engineering applications. Within this scope, bovine spinal cord tissue pieces were homogenized in 0.1 M NaOH and this viscous mixture was molded to attain 3D bioscaffolds. After resultant bioscaffolds were chemically crosslinked, the decellularization process was conducted with detergent, buffer, and enzyme solutions. Nuclear remnants in the native tissue and 3D-dCBS were determined with DNA content analysis and agarose gel electrophoresis. Afterward, 3D-dCBS were biochemically characterized in depth via glycosaminoglycan (GAG) content, hydroxyproline (HYP) assay, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cellular survival of human adipose-derived mesenchymal stem cells (hAMSCs) on the 3D-dCBS for 3rd, 7th, and 10th days was assessed via MTT assay. Scaffold and cell/scaffold constructs were also evaluated with scanning electron microscopy and histochemical studies. DNA contents for native and 3D-dCBS were respectively found to be 520.76 ± 18.11 and 28.80 ± 0.20 ng/mg dry weight (n = 3), indicating a successful decellularization process. GAG content, HYP assay, and SDS-PAGE results proved that the extracellular matrix was substantially preserved during the decellularization process. In conclusion, it is believed that the novel decellularization method may allow fabricating 3D bioscaffolds with desired geometry from soft nervous system tissues.

A Novel Protocol to Generate Decellularized Bovine Spinal Cord Extracellular Matrix-Based Scaffolds (3D-dCBS)

Extracellular matrix (ECM)-based tissue engineering scaffolds have an essential role in promoting tissue regeneration. Nerve tissue engineering aims at facilitating the repair of permanent damage to the peripheral and central nervous systems, which are difficult to heal. For this purpose, a variety of biomaterials are being developed consisting of numerous synthetic and/or natural polymers to provide axonal reinnervation and to direct the growth of axons. Here, we present a novel protocol that enables to fabricate a 3-dimensional (3D) decellularized scaffold derived from the bovine spinal cord (BSC) ECM (3D-dCBS) for neural tissue engineering applications. In this protocol, a viscous ECM-derived gel from BSC is prepared, molded, and chemically crosslinked with EDC/NHS (3D-CBS) before decellularization process. Decellularization of 3D-CBS is performed with 1% SDS to attain 3D-dCBS. As compared with other available methods, our protocol is a novel decellularization method that preserves a more significant part of the ECM. We believe that the mentioned protocol has the potential to produce a bioengineered scaffold from spinal cord tissue with desired geometry for regenerative medicine applications related to neural tissue engineering.

Age-related changes of the spinal cord: A biomechanical study.

Although it is known that aging plays an important role in the incidence and progression of cervical spondylotic myelopathy (CSM), the underlying mechanism is unclear. Studies that used fresh bovine cervical spinal cord report the gray matter of the cervical spinal cord as being more rigid and fragile than the white matter. However, there are no reports regarding the association between aging an tensile and Finite Element Method (FEM). Therefore, FEM was used based on the data pertaining to the mechanical features of older bovine cervical spinal cord to explain the pathogenesis of CSM in elderly patients. Tensile tests were conducted for white and gray matter separately in young and old bovine cervical spinal cords, and compared with their respective mechanical features. Based on the data obtained, FEM analysis was further performed, which included static and dynamic factors to describe the internal stress distribution changes of the spinal cord. These results demonstrated that the mechanical strength of young bovine spinal cords is different from that of old bovine spinal cords. The gray matter of the older spinal cord was significantly softer and more resistant to rupture compared with that of younger spinal cords (P<0.05). Among the old, although the gray matter was more fragile than the white matter, it was similar to the white matter in terms of its rigidity (P<0.05). The in vitro data were subjected to three compression patterns. The FEM analysis demonstrated that the stress level rises higher in the old spinal cords in response to similar compression, when compared with young spinal cords. These results demonstrate that in analyzing the response of the spinal cord to compression, the age of patients is an important factor to be considered, in addition to the degree of compression, compression speed and parts of the spinal cord compression factor.

Intraperitoneally administered IgG from patients with amyotrophic lateral sclerosis or from an immune-mediated goat model increase the levels of TNF-α, IL-6, and IL-10 in the spinal cord and serum of mice.

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves the selective loss of the upper and lower motor neurons (MNs). Neuroinflammation has been implicated in the pathogenesis of the sporadic form of the disease. We earlier developed immune-mediated animal models of ALS and demonstrated humoral and cellular immune reactions in the nervous system and in the sera of patients and animals. The accumulation of immunoglobulin G (IgG), an elevated intracellular level of calcium, ultrastructural alterations in the MNs, and activation of the microglia were noted in the spinal cord of ALS patients. Similar alterations developed in mice inoculated intraperitoneally with IgG from ALS patients or from an immune-mediated goat model.MethodsWe have now examined whether the intraperitoneal injection of mice with IgG from sporadic ALS patients or from immunized goats with the homogenate of the anterior horn of the bovine spinal cord is associated with changes in the pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (IL-10) cytokines in the spinal cord and serum of the mice. The levels of cytokines were measured by ELISA.ResultsIntraperitoneally administered IgG from the ALS patients induced subclinical signs of MN disease, while the injection of IgG from immunized goats resulted in a severe respiratory dysfunction and limb paralysis 24 h after the injections. Significantly increased levels of TNF-α and IL-10 were detected in the spinal cord of the mice injected with the human ALS IgG. The level of IL-6 increased primarily in the serum. The IgG from the immunized goats induced highly significant increases in the levels of all three cytokines in the serum and the spinal cord of mice.ConclusionsOur earlier experiments had proved that when ALS IgG or IgG from immune-mediated animal models was inoculated into mice, it was taken up in the MNs and had the ability to initiate damage in them. The pathological process was paralleled by microglia recruitment and activation in the spinal cord. The present experiment revealed that these forms of IgG cause significant increases in certain cytokine levels locally in the spinal cord and in the serum of the inoculated mice. These results suggest that IgG directed to the MNs may be an initial element in the damage to the MNs both in human ALS and in its immune-mediated animal models.

Molecular, dynamic, and structural origin of inhomogeneous magnetization transfer in lipid membranes

To elucidate the dynamic, structural, and molecular properties that create inhomogeneous magnetization transfer (ihMT) contrast. Amphiphilic lipids, lamellar phospholipids with cholesterol, and bovine spinal cord (BSC) specimens were examined along with nonlipid systems. Magnetization transfer (MT), enhanced MT (eMT, obtained with double-sided radiofrequency saturation), ihMT (MT - eMT), and dipolar relaxation, T1D , were measured at 2.0 and 11.7 T. The amplitude of ihMT ratio (ihMTR) is positively correlated with T1D values. Both ihMTR and T1D increase with increasing temperature in BSC white matter and in phospholipids and decrease with temperature in other lipids. Changes in ihMTR with temperature arise primarily from alterations in MT rather than eMT. Spectral width of MT, eMT, and ihMT increases with increasing carbon chain length. Concerted motions of phospholipids in white matter decrease proton spin diffusion leading to increased proton T1D times and increased ihMT amplitudes, consistent with decoupling of Zeeman and dipolar spin reservoirs. Molecular specificity and dynamic sensitivity of ihMT contrast make it a suitable candidate for probing myelin membrane disorders. Magn Reson Med 77:1318-1328, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.

Cytotoxic and inhibitory activity of ceramide on cancer cell lines

The inhibitory activity of ceramide on cancer cells was evaluated and its cytotoxicity on different cancer cell lines was measured. Ceramide was separated from bovine brain and spinal cord, and extracted by organic solvents. The crude extract was purified by using silicic acid column. Detection and identification of purified extract were carried out by using three assays: visualization, spectrophotometry and infrared. Cytotoxic effect of different concentrations (7, 15, 30 and 60 μM) of ceramide on HEp-2, RD, AMGM5, REFAM3 and AMN3 cancer cell lines was studied. Results showed that ceramide at 30 μM imposed cytotoxic activity on all cancer cell lines especially on AMGM5. Effect of ceramide at 30 μM on cell division of human lymphocyte was also examined. Significant reductions in mitotic and blast indices were observed. In addition, No genotoxic effects or chromosomal aberration were detected in lymphocyte chromosomes when ceramide was tested in vitro.

Myelin water measurement in the spinal cord

The desire to monitor the spatial-temporal characteristics of myelination in the spinal cord (SC), in the context of pathological change in demyelinating diseases or proposed neuroregenerative protocols, has led to an interest in noninvasive image-based myelin measurement methods. We present one strategy: a magnetic resonance-based measure that capitalizes on the characteristics of T(2) relaxation of water compartmentalized within tissue. In this study, 32-echo relaxation studies for measuring the myelin water fraction (MWF) were applied in healthy control SC in vivo using a sagittal inversion recovery multiecho sequence, and findings were supported with supplemental studies in bovine SC samples in vitro. Mean human MWF varied according the level of the SC examined: cervical, thoracic, and lumbar MWF was found to be 21.8 (SD=2.1)%, 24.3 (3.6)%, and 11.4 (6.4)%, respectively. Noteworthy reductions were observed in areas consistent with the expected locations of the cervical and lumbar enlargements. Average bovine MWF was 30.0 (2.7)% in white matter and 8.2 (0.4)% in gray matter. The potential applications of T(2) measurement in SC, both in characterizing disease processes like multiple sclerosis and in monitoring neuroregenerative therapies, should encourage future research in this area.

Streaming Potential of White Matter and Gray Matter in Bovine Spinal Cord under Compressive Loading

The spinal cord is mainly composed of white matter and gray matter which consist of solid and liquid phases. When the tissue deforms, the liquid phase in the tissue flows out through between the solid phases. The interaction and frictional resistance between the two phases result in the macroscopic visco-elastic behavior. In addition, the solid phase in soft tissue contains a large amount of negatively-charged proteoglycan. This electro chemical behavior affects the visco-elastic properties of the tissue. This study investigated the electro-kinetic behavior of white matter and gray matter under a variety of compressive loadings. Column-shaped specimens, 5 mm diameter and 5 mm height, were made from white matter and gray matter of bovine cervical spinal cord, and the specimens were set in physiological saline. The streaming potential was measured under compressive loading, stress relaxation, and cyclic loading. The results showed the streaming potentials to have a linear relation to tissue stress in compression and relaxation. In cyclic loading tests, the streaming potential changes according to the stress change in the loading.

The Effect of Cerebrospinal Fluid on the Biomechanics of Spinal Cord

A biomechanical study using ex vivo bovine spinal cord and dura, and a synthetic surrogate spinal cord with bovine dura. To investigate the effect of cerebrospinal fluid (CSF) on spinal cord deformation characteristics and to evaluate the biofidelity of a new surrogate spinal cord using an ex vivo bovine model of the burst fracture process. Spinal cord injury is associated with significant personal, economic and social costs. The role of CSF during the injury event and its effect on the spinal cord deformation and neurologic injury is not well understood. Such knowledge could inform preventative strategies and clinical interventions and aid the development and validation of experimental and computational models. The transverse impact of a propelled bone fragment analogue with bovine and surrogate cord models was recorded with high speed video and the images analyzed to determine deformation trajectories. Each cord specimen was tested in 3 states: with dura and CSF, with dura only, and without dura. The effect of these states on deformation magnitude, duration, and energy loss parameters was assessed. RESULTS.: The estimated spinal cord deformation was significantly reduced, although not eliminated, in the presence of CSF when compared to the bare state. The duration of deformation was generally increased in the presence of CSF, though this difference was not statistically significant. This may indicate a reduction in the cord-fragment interaction force for a given impulse. The dura was found to have no significant effect on deformation parameters for the bovine spinal cord. The deformation of the surrogate cord gave similar trends for the different states in comparison to the bovine cord, but was significantly less than the bovine spinal cord for all conditions. The results indicate that the protective mechanism of CSF may not eliminate cord deformationunder the high energy transverse impact characteristic of a burst fracture. However, CSF may contribute to a lessening of cord deformation and applied force.

Alternation of neurofilaments in immune-mediated injury of spinal cord motor neurons

Observational cross-section study. The objective of our study was to determine if phosphorylation of aggregated neurofilaments (NFs) would occur in autoimmune-mediated motor neuron injury. Our main hypothesis was that autoimmune-mediated damage of spinal cord motor neurons may influence NF phosphorylation and lead to NF aggregation. A total of 20 guinea pigs were inoculated with bovine spinal cord anterior horn homogenates (experimental autoimmune gray matter model) and 20 guinea pigs were inoculated with phosphate-buffered saline (control). NF phosphorylation and aggregation were observed by immunohistochemistry and electron microscopic examination. Data were analyzed using Student's t-test with P<0.05 being considered significant. Abnormal phosphorylation and distribution of NF occurred in motor neurons and axons of animals with experimental autoimmune gray matter disease but not in the control animals. Aberrant accumulation and phosphorylation of neurofilaments in perikarya of spinal cord motor neurons occur in immune-mediated motor neuron death. As both immunologic response and alteration of neurofilaments are observed in amyotrophic lateral sclerosis (ALS) patients and aberrant neurofilament change harms motor neurons, our present findings suggest that autoimmunity-induced ALS may mediate in part through neurofilament modification.

Fluorescence-Based Method, Exploiting Lipofuscin, for Real-Time Detection of Central Nervous System Tissues on Bovine Carcasses

The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.

Interplay between Liquid Crystalline and Isotropic Gels in Self-Assembled Neurofilament Networks

Neurofilaments (NFs) are a major constituent of nerve cell axons that assemble from three subunit proteins of low (NF-L), medium (NF-M), and high (NF-H) molecular weight into a 10 nm diameter rod with radiating sidearms to form a bottle-brush-like structure. Here, we reassemble NFs in vitro from varying weight ratios of the subunit proteins, purified from bovine spinal cord, to form homopolymers of NF-L or filaments composed of NF-L and NF-M (NF-LM), NF-L and NF-H (NF-LH), or all three subunits (NF-LMH). At high protein concentrations, NFs align to form a nematic liquid crystalline gel with a well-defined spacing determined with synchrotron small angle x-ray scattering. Near physiological conditions (86 mM monovalent salt and pH 6.8), NF-LM networks with a high NF-M grafting density favor nematic ordering whereas filaments composed of NF-LH transition to an isotropic gel at low protein concentrations as a function of increasing mole fraction of NF-H subunits. The interfilament distance decreases with NF-M grafting density, opposite the trend seen with NF-LH networks. This suggests a competition between the more attractive NF-M sidearms, forming a compact aligned nematic gel, and the repulsive NF-H sidearms, favoring a more expansive isotropic gel, at 86 mM monovalent salt. These interactions are highly salt dependent and the nematic gel phase is stabilized with increasing monovalent salt.

Identification of gangliosides recognized by IgG anti-GalNAc-GD1a antibodies in bovine spinal motor neurons and motor nerves

The presence of immunoglobulin G (IgG)-type antibodies to the ganglioside, N-acetylgalactosaminyl GD1a (GalNAc-GD1a), is closely associated with the pure motor type of Guillain–Barré syndrome (GBS). In the present study, we isolated disialogangliosides from the motor neurons and motor nerves of bovine spinal cords by DEAE-Sephadex column chromatography. The disialoganglioside fraction contained GD1a, GD2, GD1b, and three gangliosides, designated X1, X2 and X3. Serum from a patient with axonal GBS with IgG anti-GalNAc-GD1a antibody yielded positive immunostaining with X1, X2, and X3. When isolated by preparative thin-layer chromatography (TLC), X1 migrated at the same position as GalNAc-GD1a from Tay-Sachs brain, suggesting that X1 is GalNAc-GD1a containing N-acetylneuraminic acid (NeuAc). TLC of isolated X2 revealed that it migrated between GD1a and GD2. On the other hand, X3 had a migratory rate on TLC between and GD1b and GT1b. Since both X2 and X3 were recognized by IgG anti-GalNAc-GD1a antibody, the results suggest that X2 is a GalNAc-GD1a species containing a mixture containing a NeuAc-and an N-glycolylneuraminic acid (NeuGc) species, and X3 is a GalNAc-GD1a species with two NeuGc. This evidence indicating the specific localization of GalNAc-GD1a and its isomers in spinal motor neurons should be useful in elucidating the pathogenic role of IgG anti-GalNAc-GD1a antibody in pure motor-type GBS.