A Molecular Rheology Dynamics Study on 3D Printing of Liquid Crystal Elastomers.

This work presents a rheological study of a biocompatible and biodegradable liquid crystal elastomer (LCE) ink for three dimensional (3D) printing. These materials have shown that their structural variations have an effect on morphology, mechanical properties, alignment, and their impact on cell response. Within the last decade LCEs are extensively studied as potential printing materials for soft robotics applications, due to the actuation properties that are produced when liquid crystal (LC) moieties are induced through external stimuli. This report utilizes experiments and coarse-grained molecular dynamics to study the macroscopic rheology of LCEs in nonlinear shear flow. Results from the shear flow simulations are in line with the outcomes of these experimental investigations. This work believes the insights from these results can be used to design and print new material with desirable properties necessary for targeted applications.

Identifying the Edges of the Optic Cup and the Optic Disc in Glaucoma Patients by Segmentation.

With recent advancements in artificial intelligence, fundus diseases can be classified automatically for early diagnosis, and this is an interest of many researchers. The study aims to detect the edges of the optic cup and the optic disc of fundus images taken from glaucoma patients, which has further applications in the analysis of the cup-to-disc ratio (CDR). We apply a modified U-Net model architecture on various fundus datasets and use segmentation metrics to evaluate the model. We apply edge detection and dilation to post-process the segmentation and better visualize the optic cup and optic disc. Our model results are based on ORIGA, RIM-ONE v3, REFUGE, and Drishti-GS datasets. Our results show that our methodology obtains promising segmentation efficiency for CDR analysis.

Targeted Delivery of Anti-inflammatory and Imaging Agents to Microglial Cells with Polymeric Nanoparticles.

Insult to the central nervous system (CNS) results in an early inflammatory response, which can be exploited as an initial indicator of neurological dysfunction. Nanoparticle drug delivery systems provide a mechanism to increase the uptake of drugs into specific cell types in the CNS such as microglia, the resident macrophage responsible for innate immune response. In this study, we developed two nanoparticle-based carriers as potential theranostic systems for drug delivery to microglial cells. Poly(lactic-co-glycolic) acid (PLGA)- and l-tyrosine polyphosphate (LTP)-based nanoparticles were synthesized to encapsulate the magnetic resonance imaging (MRI) contrast agent, gadolinium-diethylenetriaminepentaacetic acid (Gd[DTPA]), or the anti-inflammatory drug, rolipram. Robust uptake of both polymer formulations by microglial cells was observed with no evidence of toxicity. In mixed glial cultures, we observed a preferential internalization of nanoparticles by microglia compared to that of astrocytes. Moreover, exposure of our nanoparticles to microglial cells did not induce the release of the proinflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin-1 ß (IL-1ß), or interleukin-6 (IL-6). These studies provide a foundation for the development of LTP nanoparticles as a platform for the delivery of imaging agents and drugs to the sites of neuroinflammation.

3D Porous Liquid Crystal Elastomer Foams Supporting Long-term Neuronal Cultures.

3D liquid crystal elastomer (3D-LCE) foams are used to support long-term neuronal cultures for over 60 days. Sequential imaging shows that cell density remains relatively constant throughout the culture period while the number of cells per observational area increases. In a subset of samples, retinoic acid is used to stimulate extensive neuritic outgrowth and maturation of proliferated neurons within the LCEs, inducing a threefold increase in length with cells displaying morphologies indicative of mature neurons. Designed LCEs' micro-channels have a similar diameter to endogenous parenchymal arterioles, ensuring that neurons throughout the construct have constant access to growth media during extended experiments. Here it is shown that 3D-LCEs provide a unique environment and simple method to longitudinally study spatial neuronal function, not possible in conventional culture environments, with simplistic integration into existing methodological pipelines.

Calibration and characterization of intracellular Asante Potassium Green probes, APG-2 and APG-4.

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two potassium probes, APG-4 and APG-2. APG-4 shows good sensitivity to potassium but its fluorescence is sensitive to cell volume. Because ionophores cause cell swelling, calibration buffers had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured at 132 mM and 118 mM, respectively. The other tested probe, APG-2, is nonselective for cations; this is, however, a potentially useful property because the sum [K+] + [Na+] determines the amount of intracellular water.

Staurosporine-induced apoptotic water loss is cell- and attachment-specific.

Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking. In this study, we quantified intracellular water in staurosporine-treated cells using a previously described optical microscopic technique that combines volume measurements with quantitative phase analysis. Water loss was observed in detached HeLa and in adherent MDCK but not in adherent HeLa cells. At the same time, adherent HeLa and adherent MDCK cells exhibited visually similar apoptotic morphology, including fragmentation and activation of caspase-3. Morphological changes and caspase activation were prevented by chloride channel blockers DIDS and NPPB in both adherent and suspended HeLa cells, while potassium channel blocker TEA was ineffective. We conclude that staurosporine-induced dehydration is not a universal cell response but depends on the cell type and substrate attachment and can only be judged by direct water measurements. The effects of potassium or chloride channel blockers do not always correlate with the AVD.

Changes in Methionine Metabolism and Histone H3 Trimethylation Are Linked to Mitochondrial Defects in Multiple Sclerosis.

Mitochondrial changes, including decreased expression of electron transport chain subunit genes and impaired energetic, have been reported in multiple sclerosis (MS), but the mechanisms involved in these changes are not clear. To determine whether epigenetic mechanisms are involved, we measured the concentrations of methionine metabolites by liquid chromatography tandem mass spectrometry, histone H3 methylation patterns, and markers of mitochondrial respiration in gray matter from postmortem MS and control cortical samples. We found decreases in respiratory markers as well as decreased concentrations of the methionine metabolites S-adenosylmethionine, betaine, and cystathionine in MS gray matter. We also found expression of the enzyme betaine homocysteine methyltransferase in cortical neurons. This enzyme catalyzes the remethylation of homocysteine to methionine, with betaine as the methyl donor, and has previously been thought to be restricted to liver and kidney in the adult human. Decreases in the concentration of the methyl donor betaine were correlated with decreases in histone H3 trimethylation (H3K4me3) in NeuN+ neuronal nuclei in MS cortex compared with controls. Mechanistic studies demonstrated that H3K4me3 levels and mitochondrial respiration were reduced in SH-SY5Y cells after exposure to the nitric oxide donor sodium nitroprusside, and betaine was able to rescue H3K4me3 levels and respiratory capacity in these cells. Chromatin immunoprecipitation experiments showed that betaine regulates metabolic genes in human SH-SY5Y neuroblastoma cells. These data suggest that changes to methionine metabolism may be mechanistically linked to changes in neuronal energetics in MS cortex. SIGNIFICANCE STATEMENT: For decades, it has been observed that vitamin B12 deficiency and multiple sclerosis (MS) share certain pathological changes, including conduction disturbances. In the present study, we have found that vitamin B12-dependent methionine metabolism is dysregulated in the MS brain. We found that concentrations of the methyl donor betaine are decreased in MS cortex and are correlated with reduced levels of the histone H3 methyl mark H3K4me3 in neurons. Cell culture and chromatin immunoprecipitation-seq data suggest that these changes may lead to defects in mitochondria and impact neuronal energetics. These data have uncovered a novel pathway linking methionine metabolism with mitochondrial respiration and have important implications for understanding mechanisms involved in neurodegeneration in MS.

Using stereoscopy to teach complex biological concepts.

Used effectively, stereoscopic three-dimensional (3D) technologies can engage students with complex disciplinary content as they are presented with informative representations of abstract concepts. In addition, preliminary evidence suggests that stereoscopy may enhance learning and retention in some educational settings. Biological concepts particularly benefit from this type of presentation since complex spatially oriented structures frequently define function within these systems. Viewing biological phenomena in 3D as they are in real life allows the user to relate these spatial relationships and easily grasp concepts making the key connection between structure and function. In addition, viewing these concepts interactively in 3D and in a manner that leads to increased engagement for young prospective scientists can further increase the impact. We conducted two studies evaluating the use of this technology as an instructional tool to teach high school students complex biological concepts. The first study tested the use of stereoscopic materials for teaching brain function and human anatomy to four classes. The second study evaluated stereoscopic images to support the learning of cell structure and DNA in four different high school classes. Most important, students who used stereoscopic 3D had significantly higher test scores than those who did not. In addition, students reported enjoying 3D presentations, and it was among their top choices for learning about these complex concepts. In summary, our evidence adds further support for the benefits of 3D images to students' learning of science concepts.

The First Metatarsophalangeal Joint: Updates on Revision Arthrodesis and Malunions.

First metatarsophalangeal joint (MPJ) arthrodesis procedures are a mainstay of forefoot surgery and are associated with high rates of patient satisfaction for addressing a multitude of first ray pathologic conditions. This procedure is often also used as a fallback option for the revision of poor outcomes after other surgical procedures involving the first ray. Despite its successes, there remain instances of complications that can develop after primary first MPJ arthrodesis. This article reviews first MPJ arthrodesis as a procedure for revisional surgery of the first ray, and potential surgical options after failed primary first MPJ arthrodesis.

Physical Models from Physical Templates Using Biocompatible Liquid Crystal Elastomers as Morphologically Programmable Inks For 3D Printing.

Advanced manufacturing has received considerable attention as a tool for the fabrication of cell scaffolds however, finding ideal biocompatible and biodegradable materials that fit the correct parameters for 3D printing and guide cells to align remain a challenge. Herein, a photocrosslinkable smectic-A (Sm-A) liquid crystal elastomer (LCE) designed for 3D printing is presented, that promotes cell proliferation but most importantly induces cell anisotropy. The LCE-based bio-ink allows the 3D duplication of a highly complex brain structure generated from an animal model. Vascular tissue models are generated from fluorescently stained mouse tissue spatially imaged using confocal microscopy and subsequently processed to create a digital 3D model suitable for printing. The 3D structure is reproduced using a Digital Light Processing (DLP) stereolithography (SLA) desktop 3D printer. Synchrotron Small-Angle X-ray Diffraction (SAXD) data reveal a strong alignment of the LCE layering within the struts of the printed 3D scaffold. The resultant anisotropy of the LCE struts is then shown to direct cell growth. This study offers a simple approach to produce model tissues built within hours that promote cellular alignment.

Changes to Astrocyte-associated Protein Expression at Different Timepoints of Cuprizone Treatment.

Glial cells, including astrocytes, microglia, and oligodendrocytes, are brain cells that support and dynamically interact with neurons and each other. These intercellular dynamics undergo changes during stress and disease states. In response to most forms of stress, astrocytes will undergo some variation of activation, meaning upregulation in certain proteins expressed and secreted and either upregulations or downregulations to various constitutive and normal functions. While types of activation are many and contingent on the particular disturbance that triggers these changes, there are two main overarching categories that have been delineated thus far: A1 and A2. Named in the convention of microglial activation subtypes, and with the acknowledgement that the types are not completely distinct or completely comprehensive, the A1 subtype is generically associated with toxic and pro-inflammatory factors, and the A2 phenotype is broadly associated with anti-inflammatory and neurogenic factors. The present study served to measure and document dynamic changes in these subtypes at multiple timepoints using an established experimental model of cuprizone toxic demyelination. The authors found increases in proteins associated with both cell types at different timepoints, with protein increases in the A1 marker C3d and the A2 marker Emp1 in the cortex at one week and protein increases in Emp1 in the corpus callosum at three days and four weeks. There were also increases in Emp1 staining specifically colocalized with astrocyte staining in the corpus callosum at the same timepoints as the protein increases, and in the cortex weeks later at four weeks. C3d colocalization with astrocytes also increased most at four weeks. This indicates simultaneous increases of both types of activation as well as the likely existence of astrocytes expressing both markers. The authors also found the increase in two A1 associated proteins (TNF alpha and C3d) did not show a linear relationship in line with findings from other research and indicating a more complex relationship between cuprizone toxicity and astrocyte activation. The increases in TNF alpha and IFN gamma did not occur at timepoints preceding increases in C3d and Emp1, showing that other factors also precipitate the subtypes associated (A1 for C3d and A2 for Emp1). These findings add to the body of research showing the specific early timepoints at which A1 and A2 markers are most increased during the course of cuprizone treatment, including the fact that these increases can be non-linear in the case of Emp1. This provides additional information on optimal times for targeted interventions during the cuprizone model.

Analysis of the mitochondrial proteome in multiple sclerosis cortex.

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin ß-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of MS neuropathology which result from inflammatory demyelinating events, there is another distinct mechanism involved in mitochondrial dysfunction in gray matter in MS which is not modeled in EAE.

The BHMT-betaine methylation pathway epigenetically modulates oligodendrocyte maturation.

Research into the epigenome is of growing importance as a loss of epigenetic control has been implicated in the development of neurodegenerative diseases. Previous studies have implicated aberrant DNA and histone methylation in multiple sclerosis (MS) disease pathogenesis. We have previously reported that the methyl donor betaine is depleted in MS and is linked to changes in histone H3 trimethylation (H3K4me3) in neurons. We have also shown that betaine increases histone methyltransferase activity by activating chromatin bound betaine homocysteine S-methyltransferase (BHMT). Here, we investigated the role of the BHMT-betaine methylation pathway in oligodendrocytes. Immunocytochemistry in the human MO3.13 cell line, primary rat oligodendrocytes, and tissue from MS postmortem brain confirmed the presence of the BHMT enzyme in the nucleus in oligodendrocytes. BHMT expression is increased 2-fold following oxidative insult, and qRT-PCR demonstrated that betaine can promote an increase in expression of oligodendrocyte maturation genes SOX10 and NKX-2.2 under oxidative conditions. Chromatin fractionation provided evidence of a direct interaction of BHMT on chromatin and co-IP analysis indicates an interaction between BHMT and DNMT3a. Our data show that both histone and DNA methyltransferase activity are increased following betaine administration. Betaine effects were shown to be dependent on BHMT expression following siRNA knockdown of BHMT. This is the first report of BHMT expression in oligodendrocytes and suggests that betaine acts through BHMT to modulate histone and DNA methyltransferase activity on chromatin. These data suggest that methyl donor availability can impact epigenetic changes and maturation in oligodendrocytes.

K2 Mn3 [FeII (CN)6 ]2 NPs with High T1 -Relaxivity Attributable to Water Coordination on the Mn(II) Center for Gastrointestinal Tract MR Imaging.

The lack of acid stability in the stomach and of temporal stability when moving through the gastrointestinal (GI) tract has made the development of oral magnetic resonance imaging (MRI) contrast agents based on the platform of Gd3+ -complexes problematic.On the other hand, the negative contrast enhancement produced by the T2 -weighted magnetic metal oxide nanoparticles (NPs) often renders the image readout difficult. Biocompatible NPs of the manganese Prussian blue analog K2 Mn3 [FeII (CN)6 ]2 exhibit extremely high stability under the acidic conditions of the gastric juice. Additionally, the high r1 relaxivity, low toxicity, and high temporal stability of such NPs offer great potential for the development of a true T1 -weighted oral contrast agent for MRI of the entire GI tract.

Angiotensin II-dependent growth of vascular smooth muscle cells requires transactivation of the epidermal growth factor receptor via a cytosolic phospholipase A(2)-mediated release of arachidonic acid.

Angiotensin (Ang) II stimulates vascular smooth muscle cell (VSMC) growth via activation of cytosolic phospholipase A(2) (cPLA(2)), release of arachidonic acid (ArAc) and activation of mitogen-activated protein kinase (MAPK). The mechanism linking AT(1) receptor stimulation of ArAc release with MAPK activation may involve transactivation of the epidermal growth factor receptor (EGFR). In this study, Ang II increased phosphorylation of the EGFR and MAPK in cultured VSMC and these effects were attenuated by the cPLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)), and restored by addition of ArAc. Ang II- or ArAc-induced phosphorylation of the EGFR and MAPK were abolished by the EGFR kinase inhibitor AG1478. Ang II or ArAc also stimulated VSMC growth that was blocked by AG1478 or the MAPK kinase (MEK) inhibitor PD98059. Thus, it appears that the cPLA(2)-dependent release of ArAc may provide a mechanism for the transactivation between the AT(1) receptor and the EGFR signaling cascade.

Increased blood-brain barrier hyperpermeability coincides with mast cell activation early under cuprizone administration.

The cuprizone induced animal model of demyelination is characterized by demyelination in many regions of the brain with high levels of demyelination in the corpus callosum as well as changes in neuronal function by 4-6 weeks of exposure. The model is used as a tool to study demyelination and subsequent degeneration as well as therapeutic interventions on these effects. Historically, the cuprizone model has been shown to contain no alterations to blood-brain barrier integrity, a key feature in many diseases that affect the central nervous system. Cuprizone is generally administered for 4-6 weeks to obtain maximal demyelination and degeneration. However, emerging evidence has shown that the effects of cuprizone on the brain may occur earlier than measurable gross demyelination. This study sought to investigate changes to blood-brain barrier permeability early in cuprizone administration. Results showed an increase in blood-brain barrier permeability and changes in tight junction protein expression as early as 3 days after beginning cuprizone treatment. These changes preceded glial morphological activation and demyelination known to occur during cuprizone administration. Increases in mast cell presence and activity were measured alongside the increased permeability implicating mast cells as a potential source for the blood-brain barrier disruption. These results provide further evidence of blood-brain barrier alterations in the cuprizone model and a target of therapeutic intervention in the prevention of cuprizone-induced pathology. Understanding how mast cells become activated under cuprizone and if they contribute to blood-brain barrier alterations may give further insight into how and when the blood-brain barrier is affected in CNS diseases. In summary, cuprizone administration causes an increase in blood-brain barrier permeability and this permeability coincides with mast cell activation.

Betaine restores epigenetic control and supports neuronal mitochondria in the cuprizone mouse model of multiple sclerosis.

Methionine metabolism is dysregulated in multiple sclerosis (MS). The methyl donor betaine is depleted in the MS brain where it is linked to changes in levels of histone H3 trimethylated on lysine 4 (H3K4me3) and mitochondrial impairment. We investigated the effects of replacing this depleted betaine in the cuprizone mouse model of MS. Supplementation with betaine restored epigenetic control and alleviated neurological disability in cuprizone mice. Betaine increased the methylation potential (SAM/SAH ratio), levels of H3K4me3, enhanced neuronal respiration, and prevented axonal damage. We show that the methyl donor betaine and the betaine homocysteine methyltransferase (BHMT) enzyme can act in the nucleus to repair epigenetic control and activate neuroprotective transcriptional programmes. ChIP-seq data suggest that BHMT acts on chromatin to increase the SAM/SAH ratio and histone methyltransferase activity locally to increase H3K4me3 and activate gene expression that supports neuronal energetics. These data suggest that the methyl donor betaine may provide neuroprotection in MS where mitochondrial impairment damages axons and causes disability.

Distribution of parvalbumin and calretinin immunoreactive interneurons in motor cortex from multiple sclerosis post-mortem tissue.

Parvalbumin (PV) and calretinin (CR) are calcium binding proteins (CBP's) expressed in discrete GABAergic interneuron populations in the human cortex. CBP's are known to buffer calcium concentrations and protect neurons from increases in intracellular calcium. Perturbations in intracellular calcium can activate proteolytic enzymes including calpain, leading to deleterious effects to axons. Ca++-mediated mechanisms have been found to be associated with axonal pathology in MS and the restructuring of calcium channels has been shown to occur in experimental autoimmune encephalomyelitis (EAE) as well as multiple sclerosis tissue. Previous data indicates a reduction in the expression of the parvalbumin gene as well as reduced extension of neurites on parvalbumin expressing interneurons within multiple sclerosis normal appearing grey matter (NAGM). Modifications in interneuron parvalbumin or calretinin levels could change calcium buffering capacity, as well as the way these cells respond to neuronal insults. The present study was designed to compare CBP immunoreactive neurons in normal and multiple sclerosis post-mortem NAGM. To this end, we utilized immunofluorescent staining and high resolution confocal microscopy to map regions of the human motor cortex, and characterize layer specific CBP distribution in the normal and multiple sclerosis motor cortex. Our results indicate a significant reduction in the number of PV interneurons within layer 2 of the multiple sclerosis primary motor cortex with no concurrent change in number of calretinin positive neurons.

Novel regulations of the angiotensin II receptor type 1 by calmodulin.

The angiotensin II receptor type 1 (AT1R) mediates many Ca2+-dependent actions of angiotensin II (AngII). Calmodulin (CaM) is a key transducer of Ca2+ signals in cells. Two locations on the receptor's submembrane domains (SMD) 3 and 4 are known to interact with CaM. However, the binding sites for CaM, biochemical properties of the interactions, and their functional impact are not fully understood. Using a FRET-based screening method, we identified a new binding site for CaM on SMD2 (a.a. 125-141), in addition to SMD3 and the juxtamembranous region of SMD4 (SMD4JM, a.a., 309-327). Simultaneous measurements of CaM binding and free Ca2+ show that the interactions are Ca2+-dependent, with disparate Kd and EC50(Ca2+) values within the physiological range of cytoplasmic Ca2+. Full interaction between CaM and SMD3 requires the entire domain (a.a. 215-242) and has an EC50(Ca2+) value in the range of resting cytoplasmic Ca2+, suggesting AT1R-CaM interaction can occur in resting conditions in cells. AngII induces robust ERK1/2 phosphorylation in primary vascular smooth muscle cells. This effect is suppressed by AT1R inhibitor losartan and virtually abolished by CaM antagonist W-7. AngII-induced ERK1/2 phosphorylation is suppressed in cells expressing mutant AT1R with reduced CaM binding at each identified binding domain. AngII triggers transient Ca2+ signals in cells expressing wild-type AT1R. These signals are reduced in cells expressing mutant AT1R with reduced CaM binding at SMD3 or SMD4JM, but are very slow-rising, low amplitude signal in cells expressing AT1R with reduced CaM binding at SMD2. The data indicate that CaM interactions with AT1R can occur at various domains, with different affinities, at different physiological Ca2+ levels, and are important for AT1R-mediated signaling.

Vitamin K enhances the production of brain sulfatides during remyelination.

Multiple sclerosis (MS) is a devastating neurological disease, which is characterized by multifocal demyelinating lesions in the central nervous system. The most abundant myelin lipids are galactosylceramides and their sulfated form, sulfatides, which together account for about 27% of the total dry weight of myelin. In this study we investigated the role of vitamin K in remyelination, by using an animal model for MS, the cuprizone model. Demyelination was induced in C57Bl6/J mice, by feeding them a special diet containing 0.3% cuprizone (w/w) for 6 weeks. After 6 weeks, cuprizone was removed from the diet and mice were allowed to remyelinate for either 1 or 3 weeks, in the absence or presence of vitamin K (i.p. phylloquinone, 2mg, three times per week). Vitamin K enhanced the production of total brain sulfatides, after both 1 week and 3 weeks of remyelination (n = 5, P-values were <0.0001), when compared with the control group. To determine whether or not there is a synergistic effect between vitamins K and D for the production of brain sulfatides, we employed a similar experiment as above. Vitamin K also increased the production of individual brain sulfatides, including d18:1/18:0, d18:1/20:0, d18:1/24:0, and d18:1/24:1 after 3 weeks of remyelination, when compared to the control group. In addition, vitamin D enhanced the production of total brain sulfatides, as well as d18:1/18:0, d18:1/24:0, and d18:1/24:1 sulfatides after 3 weeks of remyelination, but no synergistic effect between vitamins K and D for the production of total brain sulfatides was observed.