BACKGROUND
multiple sclerosis  is the most common disabling neurological disease of young adults; once established, it persists for the remainder of a person’s life  <cit> .
the initial triggering events which lead to ms remain unknown and there is no cure.
in ms, central nervous system  lesions form as a result of immune-mediated destruction of myelin sheaths that insulate and protect axons, resulting in loss of function and, ultimately, progressive neurodegeneration and permanent neurological decline.
current ms therapeutics mainly target the autoimmune response that damages myelin sheaths.
although effective in reducing relapses in early disease, none of them prevent long-term disease progression and none are effective in treating progressive forms of ms. a major unmet medical need in ms is the availability of therapeutics that directly protect myelin or promote new myelin formation to maintain nerve function, prevent neurodegeneration, and restore lost function in ms patients.
a key deficit in the development of myelin repair therapeutics is the lack of a high throughput axonal myelination assay suitable for drug discovery.
the closest available high throughput systems assess differentiation of purified oligodendrocyte precursor cells  or oligodendrocyte  wrapping of inert, non-biological substrates, neither of which incorporate the complex cellular interactions between opc/ols and live axons .
existing myelination assays are very low throughput, time consuming, and are designed for exploring basic research themes rather than to drive drug development decisions.
development of an assay that evaluates axonal myelination, incorporates the complete repertoire of cns cells, and can be performed at a scale and reproducibility that permits testing large numbers of potential drugs would provide a major advance in the efforts to develop remyelination-promoting therapeutics for ms patients.
we describe here the development of such an assay and utilize it to identify potential myelin repair therapeutics.
in an effort to create a myelination assay more amenable to higher throughput compound screening, we used embryonic rat cortex to develop, optimize, and validate an in vitro myelination assay  <cit>  which can be utilized for chemical library screening.
the culture system was miniaturized into a 96-well plate format enabling high throughput liquid handling, automated image acquisition and analysis of myelinating co-cultures.
it has previously been shown that inhibition of the γ-secretase protease activity promotes differentiation of opcs and myelination of retinal ganglion cells  in rgc-opc co-cultures .
based on this published work, we used the γ-secretase inhibitor , n--s-phenylglycine t-butyl ester  as a positive control in our cortical co-cultures  <cit> , and confirmed that the assay allows for the quantification of early axonal myelination in a dose-dependent manner.
this assay identifies compounds which are not active in a pure primary opc differentiation assay  <cit>  but are capable of promoting re-myelination in vivo  <cit> .
we have used this myelination assay to screen the nih clinical collection library of small molecules.
because this mixed cns cell assay platform more closely mimics the complex cellular interactions in the cns in vivo, it will also likely be scalable and adaptable to the high throughput screening of other neurological disease therapeutics in future studies.
RESULTS
development of the embryonic cortical cell co-culture assay
in an effort to move a low-throughput, well-established myelination cell culture technique to a format suitable for higher throughput screening applications, we miniaturized and automated a previously described rgc-opc co-culture technique  <cit> .
unfortunately, the low yield of rgcs and lack of assay robustness from each preparation makes this co-culture myelination assay unsuited for high-throughput compound screening .
we therefore sought another source of tissue where numbers of neurons would not be limiting.
for increased yield of primary neurons, we chose the cortex of embryonic day  <dig>  rats as an abundant source of relatively homogeneous brain cells with well-established culture methods  <cit>  .
from one litter, enough cells can easily be generated for high throughput drug screening applications .
following the differentiation and growth of neurons and glia for 5 days, we determined that the differentiation and early myelination of exogenously added opcs proceeded optimally when the growth medium was switched from nb/n <dig> to an opc-supporting myelination medium .
with this growth medium, we observed that it was not necessary to add exogenous opcs as we could readily identify mature and axon ensheathing ols that had differentiated from the embryonic cortical preparation, presumably from neural precursor cells and/or opcs.
we actually found that the differentiating opcs already present in the cultures produced better myelination .
finally, we determined that the optimal time course for myelination to proceed was 8 days after test compound addition and  <dig> div total .
figure  <dig> depicts the flow scheme of the embryonic cortical cell assay.
at this early myelination time point, we observed mbp staining aligning with smi 31/ <dig> axon staining, indicating that indeed ols are contacting and aligning with axons .fig.
<dig> flow scheme for the cortical cell myelination assay.
a dissociated cells from the cortex containing neurons and glial progenitor cells were cultured from e <dig> rat embryos onto poly-d-lysine/laminin coated 96-well plates.
b on div <dig>  when axonal projections  are apparent in the neuronal population, the growing co-culture was changed to mym media to induce ol differentiation and initiate myelination.
the following day test compounds were added and cultures were left undisturbed for an additional 8 days.
c cells were fixed and immunostained for mbp, olig <dig> and dapi on div <dig>  images were acquired using automated microscopy and scored phenotypically for myelination as described in the “methods”
fig.
<dig> oligodendrocyte processes align with cortical axons and γ-secretase inhibitors  facilitate myelination.
cortical co-cultures were treated with the gsi, dapt or dmso as described in fig.
<dig>  a on  <dig> div, cells were fixed and stained with antibodies to the axon marker smi 31/ <dig> neurofilament protein  and mbp .
image at the right is a composite of the smi 31/ <dig>  mbp, and dapi.
arrowheads indicate regions of mbp alignment with axon.
bar 100 µm.
b
left two panels show entire image fields taken from a 96-well plate immunostained for olig <dig> and mbp.
bars 200 µm.
boxed regions are enlarged in the middle panel to show morphological detail of mbp-stained ols.
bar 50 µm.
two images at right depict the digital mask of mbp staining intensity of the adjacent image  and the far right image are tracings of mbp alignment used to calculate fiber length.
bars 50 µm.
c raw data from three dapt dose response experiments was quantified from images as in b and compiled from n =  <dig> experiments,  <dig> image fields per concentration, mean ± sem.
asterisk  denotes p values versus dmso of < <dig> ; anova with bonferroni post hoc test.
there was a significant effect of four compound concentrations compared to dmso .
post hoc comparisons indicated that the mean score for the concentrations  <dig>  μm ,  <dig>  μm ,  <dig>  μm , 10 μm  was significantly different than dmso
we utilized the γ-secretase inhibitor, dapt, a known enhancer of myelination  <cit>  as a positive control to test our assay system and establish an automated morphology analysis.
after compound treatment, cells were stained for the ol lineage marker, olig <dig>  myelin basic protein  to stain mature ols, and the nuclear dye, dapi, and imaged.
myelination was scored by quantifying the characteristic change of morphology of ols when ensheathing axons—from many branched, flattened, and diffusely mbp stained processes to condensed and aligned mbp-positive fibers.
for each high resolution 10× image, we quantified the total length of contiguous, aligned mbp staining /number of olig2-positive  nuclei, referred to as myelination).
figure 2b demonstrates the digital mask created by our protocol used in the fiber length calculation.
with these methods, we determined significant dose-dependent increases in myelination with dapt .
importantly, we were able to determine reproducible ec <dig> values of four gsi compounds, dapt, ly <dig> , bms  <dig> , and mrk <dig>  allowing the ranking of compounds .
gsi-mediated facilitation of myelination was only observed in the presence of live axons and had no effect on the differentiation of purified opcs grown in isolation .
we tested two other compounds identified from published high throughput library screens that promote ol differentiation in cultures containing purified opcs, benztropine and clemastine  <cit> .
as expected, these compounds demonstrated significant ol differentiation in our acutely prepared ol differentiation assay .
however, in our cortical myelination assay, benztropine and clemastine did not promote myelination .
this data demonstrates that the cortical myelination assay identifies novel compounds with myelination activity distinct from compounds that solely promote ol differentiation.fig.
<dig> half maximal effective concentration determination of four different gsis for the promotion of myelination in the cortical culture assay.
dose response data confirm the activity of gsis and enable the calculation of the ec <dig> value for each compound.
cortical cultures were treated for 8 days with dapt, ly  <dig> , bms  <dig>  or mrk  <dig> and immunostained for mbp, olig <dig> and dapi.
dose–response curve for dapt is compiled from n =  <dig> experiments,  <dig> image fields per concentration.
representative dose–response curves for ly  <dig> , bms  <dig>  and mrk  <dig> are  <dig> image fields per concentration, mean ± sem.
respective ec <dig> values are shown in the legend
table  <dig> confirmed hits from the ncc library screen
acompounds not part of the ncc library screen
bn = ≥ 2
long-term characterization of cortical myelination cultures
to test whether the enhanced early myelination effects of gsis had longer lasting effects with the single 8 day drug treatment course, cortical cultures were treated as described followed by 2 weeks of half medium changes with fresh mym without gsi compound.
these gsi-treated cultures demonstrated robust mbp alignment compared to dmso vehicle controls .
additionally, we tested these cortical cultures for their ability to initiate the formation of axonal nodes of ranvier, essential to action potential propagation in functionally myelinated axons.
as an indicator of early node formation, we immunostained these longer term cultures with antibodies to the paranode-localized protein, caspr  <cit>  along with anti-mbp antibodies.
figure 4c demonstrates the accumulation of caspr protein at the edges of myelinated axon segments indicating that the initiation of node formation was induced by contact with ol myelin.
these data demonstrate the ability of cortical cultures to form robustly myelinated axon segments and initiate node formation which is enhanced by an early, single dose treatment with gsis.fig.
<dig> long term cortical cultures demonstrate persistent gsi-induced enhancement of myelination and initiation of axonal node of ranvier formation.
on div <dig>  cortical cultures were treated with dapt or dmso for 8 days, media was changed weekly thereafter without compound, and cells fixed on div <dig>  a
left panels show triple immunostaining of mbp , olig <dig> , and dapi .
red overlaid with blue appears pink.
right panels show digital masks created from mbp-stained images in the center panel.
masks were used for quantification of fiber length.
bars 100 µm.
arrows indicate areas with significant myelination.
b quantification of myelination showing raw data in  <dig> div cortical cultures as in a. representative data shown is averaged from  <dig> image fields per concentration, mean ± sem.
asterisk  denotes p values versus dmso of <  <dig> ; anova with dunnett’s post hoc test.
there was a significant effect of three compound concentrations compared to dmso .
post hoc comparisons indicated that the mean score for the concentrations 100 nm , 500 nm , and dapt  was significantly different than dmso.
c cortical co-cultures were grown for a total of 21 days, fixed, and immunostained for mbp  and the paranode-localized protein caspr .
note the accumulation of caspr protein at the edges of myelinated axon segments .
bar, upper panels 100 μm.
bar, lower panels 50 μm
cellular composition of cortical myelination cultures
to determine the composition of these cortical cultures, we used well-established cell type marker antibodies to identify different cell populations.
cultures were grown using the culture conditions described above, and fixed on div <dig>  all cultures were stained with the nuclear marker dapi to identify the total population of all the cells in culture.
to identify the neuronal population, we used anti-neun antibodies to identify neuronal nuclei, as well as anti-map <dig> and anti-smi 31/ <dig> neurofilament antibodies to assess the health and extent of dendrite and axon formation, respectively.
imaging of neurons in these cultures demonstrated mature cortical neurons with well-developed dendrites and a dense bed of axons .
in addition to neun for the identification of neurons, we also used anti-olig <dig> antibodies to identify opc/ols and anti-gfap antibodies to identify astrocytes.
we then quantified the percentage of each of these cell types in this cortical co-culture preparation as a percentage of the total cell population identified with dapi nuclear staining of all cells.
we found that the cell composition under these culture conditions was 23 % neurons, 46 % astrocytes, 22 % opcs/ols, and 9 % unidentified cells.
in order to better understand how ols differentiate and develop in these cultures, we stained and imaged div <dig> cultures to assess the cell composition of our cultures on the day of test compound addition.
at this stage, the cultures contained ~50 % neurons, having already generated an axon network .
since our cultures were derived from embryonic cortex, we used the bi-potent o2a glial progenitor antibody marker a2b <dig>  <cit> , to identify glial progenitors still capable of differentiating.
div <dig> cultures contained abundant a2b <dig> positive cells which were not observed at div <dig> .
there were relatively few astrocytes  and differentiated ols  at this stage, indicating that a majority of the ol differentiation and myelination occurred during the test compound treatment window .
using the microglial marker iba <dig>  we detected <1 % microglial cells at div <dig>  and undetectable microglia at div <dig>
nih clinical collection library screening for compounds that promote myelination
to demonstrate that the assay conditions we developed were robust enough to support drug discovery screening efforts, we screened a small library of compounds.
we selected the nih clinical collection  library for screening which contains food and drug administration -approved off-patent drugs.
therefore, hits retrieved from this collection could lead to potential drug candidates for further development and rapid repositioning as therapeutics for ms. the ncc library consists of  <dig> biologically active compounds that have been through phase i–iii clinical trials.
this collection is additionally attractive because of the wide variety of cellular targets that are represented.
because this focused fda-approved compound collection is small and the drug structures diverse, we decided to screen at two concentrations  to reduce the possibility of missing hits due to false negatives.
each plate contained eight wells treated with dmso or dapt controls and each test compound concentration was screened in duplicate.
automated image acquisition was performed from four randomized fields from each well, representing a total of eight data points per test concentration.
the data was analyzed to find active compounds that increase myelin formation above a pre-defined threshold .
though the threshold is somewhat arbitrary, we included in our analysis the delineation of three sds above the mean signal of dmso-treated well .
while not a criterion in our assay for hit selection, it provided a statistical assurance that we were well out of a false-positive hit rate  range.
control dapt versus dmso values from the entire myelination screen were highly statistically significant  indicating an acceptable screen window.fig.
<dig> analysis of the cortical myelination screen of the ncc compound library.
a high-throughput screening data set used to identify promoters of myelination.
the mean response is indicated by the solid line.
the dotted line delineates the value of three sds above the mean.
compounds that significantly reduced olig <dig> expression were excluded.
b high-throughput screening data plate control values of myelination.
each point is an compiled control value from each screening plate .
c using the fiber score/mbp score as a specific measure of myelination , the ratio of the dapt to dmso controls demonstrates the screening assay window.
the red line delineates the cutoff value of  <dig> .
each point is an averaged value from each screening plate .
the average dapt/dmso-fiber score/mbp score for the entire ncc library screen =  <dig>  .
d ncc library hit selection process in the cortical culture myelination assay.
fifty-three primary hits compounds were initially identified from the ncc library with the criteria of >50 % dapt and > <dig>  fiber score/mbp score or fiber/mbp.
the primary hits were further refined with additional criteria of >25 % dapt/olig <dig> nuclei ratio, <40 % dapi/olig <dig> nuclei ratio, and a visual morphology check to yield refined hits of  <dig> compounds.
all refined hit compounds were reordered fresh and tested for efficacy in a dose–response profile.
ten compounds passed these criteria and were confirmed as hits
we were concerned about the possibility of inter-preparation variability and differences in the behaviors of dissociated primary neurons and glia in culture, so we considered how to evaluate the consistency of responses to treatment in addition to assessing the consistency of the plate controls.
as a measure of assay quality, we considered the z-factor; however, since our assay is based on multiple readouts, we incorporated an additional parameter for assay window and robustness, a specific morphological measurement of early myelination.
this criterion is a quotient of two morphological measurements—fiber length intensity score and mbp intensity score which adjusts for the contribution of ol differentiation  .
a ratio of one indicates that an observed increase in myelination may almost be entirely accounted for by an increase in the extent of ol differentiation, whereas a value significantly greater than one indicates that there is an observed increase in myelination above and beyond what would be expected by an increase in ol differentiation alone, i.e.
specific induction of myelination.
we primarily used this to measure the quality of each plate in the screen.
we typically observed dapt treated cultures to have fiber score/mbp scores > <dig> .
figure 5c depicts the dapt/dmso fiber score/mbp scores of each plate from the entire library screen which generated an average fiber score/mbp score of  <dig> .
for each plate in the screen, the acceptable fiber score/mbp score cutoff was in the range of  <dig>  ±  <dig> .
in addition, the fiber score/mbp score was incorporated into the criteria for assay hits to potentially distinguish between active compounds with distinct mechanisms of action .
additional file 12: table s <dig> contains the data set for the entire library screen, showing the % dapt normalized scores for myelination for each library compound.
selection criteria for myelination assay positive hit compounds
to select candidate compounds with substantial activity in our screen, compounds that had scores >50 % of dapt were included as secondary hits .
in addition, to further delineate the myelination effect enhanced by compounds, we implemented a second criterion of fiber score/mbp score  including any compounds that had scores > <dig>  .
three compounds passed this criterion alone and were included in further hit compound refinement.
we observed that some compounds displayed unusually large myelination scores but had very low overall olig2+ nuclei expression, which could either reflect an inhibition of ol proliferation or increased ol cell death .
not surprisingly, visual assessment of images from these cultures revealed a low overall number of myelinating ols.
we therefore incorporated a third criterion to eliminate compounds that primarily act as anti-proliferative compounds and thus greatly reduced olig <dig> expression: the ratio of total nuclei /olig2+ nuclei.
large dapi/olig2+ nuclei numbers  were an indicator that the test compound severely depleted opcs and ols, undesirable in a screen for compounds that promote myelination.
dapt reduces the number of olig2+ cells by ~50 %, most likely by promoting opc differentiation and reducing opc proliferation  <cit> .
therefore, we implemented a criterion of >25 % of the dapt olig2+ cell count which also effectively eliminated compounds that severely reduced the number of olig2+ cells .
a fourth criterion was the qualitative assessment of ol mbp staining, taking into account the number of ols/image field and ol morphology.
compounds that dramatically changed ol morphology  while reducing the number of ols/field were eliminated.
active compounds that passed all of these criteria were referred to as refined hits .
a fifth criterion was to confirm activity and potency of refined hit compounds with full dose–response curve experiments of at least two replicates using reordered or resynthesized material.
actives that met this criterion were referred to as our confirmed hits  and our hit rate is based on this number.
in a screen of  <dig> fda-approved drugs, our screen identified  <dig> primary hits,  <dig> refined secondary hits and ten confirmed and reproducible hits .
the resulting hit rate for the entire screen was ~ <dig>  %.
additional file 11: figure s <dig> shows the chemical structure of each hit compound, screening image of mbp/olig2/dapi staining, and the ec <dig> curves for myelination.
table  <dig> shows the calculated myelination ec <dig> values for the top hits from our cortical myelination screen.
based on the available literature on these previously characterized compounds, we grouped the hits based on the known mechanisms of action.
these compounds fell into many different classes, grouped in table  <dig>  and are distinct from compounds previously identified by other library screens that have used ol differentiation assays in the absence of axons .
these data represent an initial screen to identify myelination-promoting compounds.
future studies will include an investigation of compound mechanisms as well as a thorough characterization of how these compounds effect cellular composition of the cultures.
discussion
development of high throughput cortical myelination assay
it has become clear that focusing solely on the immunological component of ms only addresses one aspect of the disease.
repairing damaged myelin and/or promoting the remyelination of demyelinated axons within lesions would, at a minimum, facilitate the preservation and/or restoration of some neuronal function.
this may also prevent the irreversible neuronal damage believed to underlie the progressive disability that eventually affects most ms patients.
thus, remyelinating compounds are highly sought after, but have been difficult to identify in part because of the lack of hts assays that truly detect myelination.
our goal for developing a co-culture with live axons and oligodendrocytes as a myelinating in vitro system was to overcome the challenges of labor intensive opc/neuron preparations, inconsistent performance of classical sources of neurons for modeling myelination , and generating sufficient quantities of cells required for a robust hts assay.
in this study, we have described the development of the first in vitro myelination assay that assesses the functional dynamic interaction between live axons and oligodendrocytes during myelination and can be performed with a simple culture technique at a scale and reproducibility amenable to hts drug discovery.
this assay is unique in that it evaluates test compounds in the presence of the co-developing milieu of native brain cells, including ols, neurons, and astrocytes.
we demonstrate here that primary embryonic cortical tissue is an abundant cell source for both neurons and opcs that are myelination competent  <cit> , easier to culture than rgcs, and widely used in large-scale hts screening within the pharmaceutical industry.
we have validated this assay using gsis, establishing ec <dig> values for four different compounds to allow the ranking of potency.
using this assay as an initial screen, we screened the ncc library and identified ten confirmed hit compounds from diverse target classes for follow-up characterization.
perhaps the most important aspects of the cortical myelination assay are that ols develop and differentiate alongside growing axons and astrocytes, two major sources of signaling molecules known to influence myelination.
the expression of the axonal protein lrr and ig domain-containing, nogo receptor-interacting protein  was demonstrated be a potent inhibitor of differentiation and myelination .
indeed, anti-lingo- <dig> antibody is being developed as an ms therapeutic to promote axon remyelination and is currently in human clinical trials .
leukemia inhibitory factor  has been shown to be released by astrocytes in response to atp from action potential firing axons to promote myelination  <cit> .
additionally, through the action of tnfr <dig> on astrocytes, lif is produced to stimulate ol differentiation in a co-culture system  <cit> .
furthermore, astrocytes were demonstrated to reduce ol differentiation, but specifically enhance myelin thickness and the rate of axon wrapping  <cit> .
tnf impairs ol differentiation  <cit>  attenuating tnf signaling by tnfr <dig> blocking therapy ameliorates ms symptoms in eae  <cit> .
it was also demonstrated that inhibiting glial γ-secretase promoted myelination  <cit> , which also showed efficacy in vivo in the eae model of ms  <cit> .
these observations emphasize the importance of having culture conditions that more closely mimic the in vivo cns composition.
it is important to note that this cortical myelination assay may fail to identify all possible compounds that may promote myelination.
due to the relatively short time course and single compound addition, our assay may not identify compounds with short half-lives, require multiple target activations, or require longer time courses.
the assay could be modified by spiking in test compounds during the course of the ensheathment window and/or lengthen the ensheathment window longer than 8 days.
we included t <dig>  forskolin, and cntf in the mym medium as factors that facilitate ol differentiation and survival  <cit> .
the activity of these factors may mask effects of potential stimulators of myelination.
in particular, elimination of t <dig>  may lower the threshold for identifying additional candidate compounds.
this stimulation of differentiation by t <dig> may account for the lack of ol differentiation activity of benztropine and clemastine in our cortical myelination assay.
elimination of these factors from the myelination phase of the assay may reveal additional compounds with myelination activity.
the cortical myelination assay identifies compounds not revealed by ol differentiation assays
our myelination assay greatly differs from in vitro ol differentiation assays used for compound screening which have only assessed differentiation using purified opcs  adapted to culture conditions by multiple passages  <cit> , or differentiated from induced pluripotent stem cells  <cit>  and carried out in very short developmental time frames.
mei et al,  <dig>  <cit>  developed an hts assay incorporating ol differentiation in the presence of inert micropillers allowing the quantification of pillar wrapping as a surrogate for myelination  <cit> .
lead compounds identified from these three studies, including clemastine, benztropine, miconazole and clobetasol, facilitate ol differentiation in cultures of purified opcs  , but had no effect on myelination in our live axon myelination assay .
notably, our myelination assay did not identify muscarinic antagonists as previously identified by independent ol differentiation screens  <cit> , but revealed entirely new classes of compounds that promote myelination.
no other high throughput assay has been developed to date capable of assessing the initiation and facilitation of myelination in the presence of axons, arguably the most important features when selecting candidate compounds capable of promoting or generating remyelination.
relation of cortical myelination assay hit compounds to clinical applications and multiple sclerosis
repositioning approved drugs for the treatment of new indications is an activity that has grown in popularity in recent years and is a trend that is predicted to continue.
there are many examples where repurposed drugs are making an impact in the field of ms. a few to note are bg- <dig> , a modification of fumaderm®, used to treat psoriasis; fingolimod , originally developed for transplantation and alemtuzumab , an oncology drug.
we found that eight out of our ten confirmed hit compounds aligned with current ms repositioning efforts and are briefly cited here.
a treatment for congestive heart failure, digoxin  enhances both myelination and differentiation in our assay.
it has been shown to reverse conduction block in demyelinated nerve fibers in experimental animals.
moreover, digoxin and possibly its derivatives, have also been implicated as a possible therapeutic for ms. a clinical study involving ms patients, showed improvement of symptoms when given the drug intravenously  <cit> .
other hits have shown efficacy in ms animal models.
imatinib mesylate , a kinase inhibitor used for cancer treatment, is efficacious in experimental autoimmune encephalomyelitis  models  <cit> .
artesunate, a natural compound used to treat malaria, has a derivative form  that has anti-inflammatory properties in eae  <cit> .
methotrexate , an anti-metabolite used to treat rheumatoid arthritis, is also efficacious at restoring myelin in cuprizone-induced demyelination mouse models  <cit> .
in addition, methotrexate is commonly used off-label in ms, and is suggested as a low-dose, add-on therapy in combination with other treatments, to reduce the relapsing-remitting rate in ms  <cit>  and to slow the progression of ms.
some hits have been evaluated or will soon be tested in human ms patients.
oxcarbazepine , a treatment for seizures and amilioride , a treatment for high blood pressure, have been demonstrated to be neuroprotective in rodent models of ms and in ms patients  <cit> , and are now in human clinical trials to treat progressive ms .
additionally, there is a patent for docetaxel , an anti-microtubule and treatment for cancer  on its use to treat or prevent psoriasis and ms.
off-label muscle relaxants are used, when necessary, to help ms patients offset or reduce relapsing-remitting episodes.
atracurium besylate  is used as an adjunct to general anesthesia and particularly useful for ms patients undergoing surgery since general anesthesia can exacerbate ms symptoms.
in a clinical case report, a patient with ms underwent surgery for renal failure, and in a 3 month follow-up, reported no further ms attacks  <cit> .
the remainder of our confirmed hits, zu-capsaicin  and tegafur  have not been tested in any demyelinating diseases.
to our knowledge the myelin restorative effects of these compounds have not been evaluated.
our data suggests that these drugs should additionally focus on testing these hits to determine whether they have beneficial effects on remyelination in vivo and possibly in ms patients.
CONCLUSIONS
we have developed the first high capacity, phenotypic in vitro assay amenable to high throughput screening to identify compounds that promote myelination.
our myelination assay incorporates both live axons and astrocytes, both of which have profound effects on myelination in vivo.
our assay utilizes primary cortical cells under conditions that allows for endogenous myelination, and thus avoids potential artefacts due to the use of cell lines or artificial substrates for myelination.
to demonstrate the utility of this assay, we have screened the ncc library of fda-approved compounds, identifying ten compounds for follow up studies, including effects on different cell types, mechanisms of action, structure–activity relationship determination, and in in vivo models of ms. our hope is that this cortical myelination assay will be scaled up to screen even larger compound libraries in search of more efficacious ms therapeutics.

 multiple sclerosis is the most common disabling neurological disease of young adults; once established, it persists for the remainder of a person’s life. the initial triggering events which lead to ms remain unknown and there is no cure. in ms, central nervous system lesions form as a result of immune-mediated destruction of myelin sheaths that insulate and protect axons, resulting in loss of function and, ultimately, progressive neurodegeneration and permanent neurological decline. current ms therapeutics mainly target the autoimmune response that damages myelin sheaths. although effective in reducing relapses in early disease, none of them prevent long-term disease progression and none are effective in treating progressive forms of ms. a major unmet medical need in ms is the availability of therapeutics that directly protect myelin or promote new myelin formation to maintain nerve function, prevent neurodegeneration, and restore lost function in ms patients. a key deficit in the development of myelin repair therapeutics is the lack of a high throughput axonal myelination assay suitable for drug discovery. the closest available high throughput systems assess differentiation of purified oligodendrocyte precursor cells or oligodendrocyte wrapping of inert, non-biological substrates, neither of which incorporate the complex cellular interactions between opc/ols and live axons. existing myelination assays are very low throughput, time consuming, and are designed for exploring basic research themes rather than to drive drug development decisions. development of an assay that evaluates axonal myelination, incorporates the complete repertoire of cns cells, and can be performed at a scale and reproducibility that permits testing large numbers of potential drugs would provide a major advance in the efforts to develop remyelination-promoting therapeutics for ms patients. we describe here the development of such an assay and utilize it to identify potential myelin repair therapeutics. in an effort to create a myelination assay more amenable to higher throughput compound screening, we used embryonic rat cortex to develop, optimize, and validate an in vitro myelination assay which can be utilized for chemical library screening. the culture system was miniaturized into a 96-well plate format enabling high throughput liquid handling, automated image acquisition and analysis of myelinating co-cultures. it has previously been shown that inhibition of the γ-secretase protease activity promotes differentiation of opcs and myelination of retinal ganglion cells in rgc-opc co-cultures. based on this published work, we used the γ-secretase inhibitor, n--s-phenylglycine t-butyl ester as a positive control in our cortical co-cultures, and confirmed that the assay allows for the quantification of early axonal myelination in a dose-dependent manner. this assay identifies compounds which are not active in a pure primary opc differentiation assay but are capable of promoting re-myelination in vivo. we have used this myelination assay to screen the nih clinical collection library of small molecules. because this mixed cns cell assay platform more closely mimics the complex cellular interactions in the cns in vivo, it will also likely be scalable and adaptable to the high throughput screening of other neurological disease therapeutics in future studies.  development of the embryonic cortical cell co-culture assay in an effort to move a low-throughput, well-established myelination cell culture technique to a format suitable for higher throughput screening applications, we miniaturized and automated a previously described rgc-opc co-culture technique. unfortunately, the low yield of rgcs and lack of assay robustness from each preparation makes this co-culture myelination assay unsuited for high-throughput compound screening. we therefore sought another source of tissue where numbers of neurons would not be limiting. for increased yield of primary neurons, we chose the cortex of embryonic day rats as an abundant source of relatively homogeneous brain cells with well-established culture methods. from one litter, enough cells can easily be generated for high throughput drug screening applications. following the differentiation and growth of neurons and glia for 5 days, we determined that the differentiation and early myelination of exogenously added opcs proceeded optimally when the growth medium was switched from nb/n to an opc-supporting myelination medium. with this growth medium, we observed that it was not necessary to add exogenous opcs as we could readily identify mature and axon ensheathing ols that had differentiated from the embryonic cortical preparation, presumably from neural precursor cells and/or opcs. we actually found that the differentiating opcs already present in the cultures produced better myelination. finally, we determined that the optimal time course for myelination to proceed was 8 days after test compound addition and div total. figure  depicts the flow scheme of the embryonic cortical cell assay. at this early myelination time point, we observed mbp staining aligning with smi 31/ axon staining, indicating that indeed ols are contacting and aligning with axons.fig.  flow scheme for the cortical cell myelination assay. a dissociated cells from the cortex containing neurons and glial progenitor cells were cultured from e rat embryos onto poly-d-lysine/laminin coated 96-well plates. b on div when axonal projections are apparent in the neuronal population, the growing co-culture was changed to mym media to induce ol differentiation and initiate myelination. the following day test compounds were added and cultures were left undisturbed for an additional 8 days. c cells were fixed and immunostained for mbp, olig and dapi on div images were acquired using automated microscopy and scored phenotypically for myelination as described in the “methods” fig.  oligodendrocyte processes align with cortical axons and γ-secretase inhibitors facilitate myelination. cortical co-cultures were treated with the gsi, dapt or dmso as described in fig.  a on div, cells were fixed and stained with antibodies to the axon marker smi 31/ neurofilament protein and mbp. image at the right is a composite of the smi 31/ mbp, and dapi. arrowheads indicate regions of mbp alignment with axon. bar 100 µm. b left two panels show entire image fields taken from a 96-well plate immunostained for olig and mbp. bars 200 µm. boxed regions are enlarged in the middle panel to show morphological detail of mbp-stained ols. bar 50 µm. two images at right depict the digital mask of mbp staining intensity of the adjacent image and the far right image are tracings of mbp alignment used to calculate fiber length. bars 50 µm. c raw data from three dapt dose response experiments was quantified from images as in b and compiled from n =  experiments, image fields per concentration, mean ± sem. asterisk denotes p values versus dmso of <; anova with bonferroni post hoc test. there was a significant effect of four compound concentrations compared to dmso. post hoc comparisons indicated that the mean score for the concentrations  μm,  μm,  μm, 10 μm was significantly different than dmso we utilized the γ-secretase inhibitor, dapt, a known enhancer of myelination as a positive control to test our assay system and establish an automated morphology analysis. after compound treatment, cells were stained for the ol lineage marker, olig myelin basic protein to stain mature ols, and the nuclear dye, dapi, and imaged. myelination was scored by quantifying the characteristic change of morphology of ols when ensheathing axons—from many branched, flattened, and diffusely mbp stained processes to condensed and aligned mbp-positive fibers. for each high resolution 10× image, we quantified the total length of contiguous, aligned mbp staining /number of olig2-positive nuclei, referred to as myelination). figure 2b demonstrates the digital mask created by our protocol used in the fiber length calculation. with these methods, we determined significant dose-dependent increases in myelination with dapt. importantly, we were able to determine reproducible ec values of four gsi compounds, dapt, ly, bms, and mrk allowing the ranking of compounds. gsi-mediated facilitation of myelination was only observed in the presence of live axons and had no effect on the differentiation of purified opcs grown in isolation. we tested two other compounds identified from published high throughput library screens that promote ol differentiation in cultures containing purified opcs, benztropine and clemastine. as expected, these compounds demonstrated significant ol differentiation in our acutely prepared ol differentiation assay. however, in our cortical myelination assay, benztropine and clemastine did not promote myelination. this data demonstrates that the cortical myelination assay identifies novel compounds with myelination activity distinct from compounds that solely promote ol differentiation.fig.  half maximal effective concentration determination of four different gsis for the promotion of myelination in the cortical culture assay. dose response data confirm the activity of gsis and enable the calculation of the ec value for each compound. cortical cultures were treated for 8 days with dapt, ly, bms or mrk and immunostained for mbp, olig and dapi. dose–response curve for dapt is compiled from n =  experiments, image fields per concentration. representative dose–response curves for ly, bms and mrk are image fields per concentration, mean ± sem. respective ec values are shown in the legend table  confirmed hits from the ncc library screen acompounds not part of the ncc library screen bn = ≥ 2 long-term characterization of cortical myelination cultures to test whether the enhanced early myelination effects of gsis had longer lasting effects with the single 8 day drug treatment course, cortical cultures were treated as described followed by 2 weeks of half medium changes with fresh mym without gsi compound. these gsi-treated cultures demonstrated robust mbp alignment compared to dmso vehicle controls. additionally, we tested these cortical cultures for their ability to initiate the formation of axonal nodes of ranvier, essential to action potential propagation in functionally myelinated axons. as an indicator of early node formation, we immunostained these longer term cultures with antibodies to the paranode-localized protein, caspr along with anti-mbp antibodies. figure 4c demonstrates the accumulation of caspr protein at the edges of myelinated axon segments indicating that the initiation of node formation was induced by contact with ol myelin. these data demonstrate the ability of cortical cultures to form robustly myelinated axon segments and initiate node formation which is enhanced by an early, single dose treatment with gsis.fig.  long term cortical cultures demonstrate persistent gsi-induced enhancement of myelination and initiation of axonal node of ranvier formation. on div cortical cultures were treated with dapt or dmso for 8 days, media was changed weekly thereafter without compound, and cells fixed on div a left panels show triple immunostaining of mbp, olig, and dapi. red overlaid with blue appears pink. right panels show digital masks created from mbp-stained images in the center panel. masks were used for quantification of fiber length. bars 100 µm. arrows indicate areas with significant myelination. b quantification of myelination showing raw data in div cortical cultures as in a. representative data shown is averaged from image fields per concentration, mean ± sem. asterisk denotes p values versus dmso of < ; anova with dunnett’s post hoc test. there was a significant effect of three compound concentrations compared to dmso. post hoc comparisons indicated that the mean score for the concentrations 100 nm, 500 nm, and dapt was significantly different than dmso. c cortical co-cultures were grown for a total of 21 days, fixed, and immunostained for mbp and the paranode-localized protein caspr. note the accumulation of caspr protein at the edges of myelinated axon segments. bar, upper panels 100 μm. bar, lower panels 50 μm cellular composition of cortical myelination cultures to determine the composition of these cortical cultures, we used well-established cell type marker antibodies to identify different cell populations. cultures were grown using the culture conditions described above, and fixed on div all cultures were stained with the nuclear marker dapi to identify the total population of all the cells in culture. to identify the neuronal population, we used anti-neun antibodies to identify neuronal nuclei, as well as anti-map and anti-smi 31/ neurofilament antibodies to assess the health and extent of dendrite and axon formation, respectively. imaging of neurons in these cultures demonstrated mature cortical neurons with well-developed dendrites and a dense bed of axons. in addition to neun for the identification of neurons, we also used anti-olig antibodies to identify opc/ols and anti-gfap antibodies to identify astrocytes. we then quantified the percentage of each of these cell types in this cortical co-culture preparation as a percentage of the total cell population identified with dapi nuclear staining of all cells. we found that the cell composition under these culture conditions was 23 % neurons, 46 % astrocytes, 22 % opcs/ols, and 9 % unidentified cells. in order to better understand how ols differentiate and develop in these cultures, we stained and imaged div cultures to assess the cell composition of our cultures on the day of test compound addition. at this stage, the cultures contained ~50 % neurons, having already generated an axon network. since our cultures were derived from embryonic cortex, we used the bi-potent o2a glial progenitor antibody marker a2b, to identify glial progenitors still capable of differentiating. div cultures contained abundant a2b positive cells which were not observed at div. there were relatively few astrocytes and differentiated ols at this stage, indicating that a majority of the ol differentiation and myelination occurred during the test compound treatment window. using the microglial marker iba we detected <1 % microglial cells at div and undetectable microglia at div nih clinical collection library screening for compounds that promote myelination to demonstrate that the assay conditions we developed were robust enough to support drug discovery screening efforts, we screened a small library of compounds. we selected the nih clinical collection library for screening which contains food and drug administration -approved off-patent drugs. therefore, hits retrieved from this collection could lead to potential drug candidates for further development and rapid repositioning as therapeutics for ms. the ncc library consists of biologically active compounds that have been through phase i–iii clinical trials. this collection is additionally attractive because of the wide variety of cellular targets that are represented. because this focused fda-approved compound collection is small and the drug structures diverse, we decided to screen at two concentrations to reduce the possibility of missing hits due to false negatives. each plate contained eight wells treated with dmso or dapt controls and each test compound concentration was screened in duplicate. automated image acquisition was performed from four randomized fields from each well, representing a total of eight data points per test concentration. the data was analyzed to find active compounds that increase myelin formation above a pre-defined threshold. though the threshold is somewhat arbitrary, we included in our analysis the delineation of three sds above the mean signal of dmso-treated well. while not a criterion in our assay for hit selection, it provided a statistical assurance that we were well out of a false-positive hit rate range. control dapt versus dmso values from the entire myelination screen were highly statistically significant indicating an acceptable screen window.fig.  analysis of the cortical myelination screen of the ncc compound library. a high-throughput screening data set used to identify promoters of myelination. the mean response is indicated by the solid line. the dotted line delineates the value of three sds above the mean. compounds that significantly reduced olig expression were excluded. b high-throughput screening data plate control values of myelination. each point is an compiled control value from each screening plate. c using the fiber score/mbp score as a specific measure of myelination, the ratio of the dapt to dmso controls demonstrates the screening assay window. the red line delineates the cutoff value of. each point is an averaged value from each screening plate. the average dapt/dmso-fiber score/mbp score for the entire ncc library screen = . d ncc library hit selection process in the cortical culture myelination assay. fifty-three primary hits compounds were initially identified from the ncc library with the criteria of >50 % dapt and > fiber score/mbp score or fiber/mbp. the primary hits were further refined with additional criteria of >25 % dapt/olig nuclei ratio, <40 % dapi/olig nuclei ratio, and a visual morphology check to yield refined hits of compounds. all refined hit compounds were reordered fresh and tested for efficacy in a dose–response profile. ten compounds passed these criteria and were confirmed as hits we were concerned about the possibility of inter-preparation variability and differences in the behaviors of dissociated primary neurons and glia in culture, so we considered how to evaluate the consistency of responses to treatment in addition to assessing the consistency of the plate controls. as a measure of assay quality, we considered the z-factor; however, since our assay is based on multiple readouts, we incorporated an additional parameter for assay window and robustness, a specific morphological measurement of early myelination. this criterion is a quotient of two morphological measurements—fiber length intensity score and mbp intensity score which adjusts for the contribution of ol differentiation. a ratio of one indicates that an observed increase in myelination may almost be entirely accounted for by an increase in the extent of ol differentiation, whereas a value significantly greater than one indicates that there is an observed increase in myelination above and beyond what would be expected by an increase in ol differentiation alone, i.e. specific induction of myelination. we primarily used this to measure the quality of each plate in the screen. we typically observed dapt treated cultures to have fiber score/mbp scores >. figure 5c depicts the dapt/dmso fiber score/mbp scores of each plate from the entire library screen which generated an average fiber score/mbp score of. for each plate in the screen, the acceptable fiber score/mbp score cutoff was in the range of  ± . in addition, the fiber score/mbp score was incorporated into the criteria for assay hits to potentially distinguish between active compounds with distinct mechanisms of action. additional file 12: table s contains the data set for the entire library screen, showing the % dapt normalized scores for myelination for each library compound. selection criteria for myelination assay positive hit compounds to select candidate compounds with substantial activity in our screen, compounds that had scores >50 % of dapt were included as secondary hits. in addition, to further delineate the myelination effect enhanced by compounds, we implemented a second criterion of fiber score/mbp score including any compounds that had scores >. three compounds passed this criterion alone and were included in further hit compound refinement. we observed that some compounds displayed unusually large myelination scores but had very low overall olig2+ nuclei expression, which could either reflect an inhibition of ol proliferation or increased ol cell death. not surprisingly, visual assessment of images from these cultures revealed a low overall number of myelinating ols. we therefore incorporated a third criterion to eliminate compounds that primarily act as anti-proliferative compounds and thus greatly reduced olig expression: the ratio of total nuclei /olig2+ nuclei. large dapi/olig2+ nuclei numbers were an indicator that the test compound severely depleted opcs and ols, undesirable in a screen for compounds that promote myelination. dapt reduces the number of olig2+ cells by ~50 %, most likely by promoting opc differentiation and reducing opc proliferation. therefore, we implemented a criterion of >25 % of the dapt olig2+ cell count which also effectively eliminated compounds that severely reduced the number of olig2+ cells. a fourth criterion was the qualitative assessment of ol mbp staining, taking into account the number of ols/image field and ol morphology. compounds that dramatically changed ol morphology while reducing the number of ols/field were eliminated. active compounds that passed all of these criteria were referred to as refined hits. a fifth criterion was to confirm activity and potency of refined hit compounds with full dose–response curve experiments of at least two replicates using reordered or resynthesized material. actives that met this criterion were referred to as our confirmed hits and our hit rate is based on this number. in a screen of fda-approved drugs, our screen identified primary hits, refined secondary hits and ten confirmed and reproducible hits. the resulting hit rate for the entire screen was ~  %. additional file 11: figure s shows the chemical structure of each hit compound, screening image of mbp/olig2/dapi staining, and the ec curves for myelination. table  shows the calculated myelination ec values for the top hits from our cortical myelination screen. based on the available literature on these previously characterized compounds, we grouped the hits based on the known mechanisms of action. these compounds fell into many different classes, grouped in table  and are distinct from compounds previously identified by other library screens that have used ol differentiation assays in the absence of axons. these data represent an initial screen to identify myelination-promoting compounds. future studies will include an investigation of compound mechanisms as well as a thorough characterization of how these compounds effect cellular composition of the cultures. discussion development of high throughput cortical myelination assay it has become clear that focusing solely on the immunological component of ms only addresses one aspect of the disease. repairing damaged myelin and/or promoting the remyelination of demyelinated axons within lesions would, at a minimum, facilitate the preservation and/or restoration of some neuronal function. this may also prevent the irreversible neuronal damage believed to underlie the progressive disability that eventually affects most ms patients. thus, remyelinating compounds are highly sought after, but have been difficult to identify in part because of the lack of hts assays that truly detect myelination. our goal for developing a co-culture with live axons and oligodendrocytes as a myelinating in vitro system was to overcome the challenges of labor intensive opc/neuron preparations, inconsistent performance of classical sources of neurons for modeling myelination, and generating sufficient quantities of cells required for a robust hts assay. in this study, we have described the development of the first in vitro myelination assay that assesses the functional dynamic interaction between live axons and oligodendrocytes during myelination and can be performed with a simple culture technique at a scale and reproducibility amenable to hts drug discovery. this assay is unique in that it evaluates test compounds in the presence of the co-developing milieu of native brain cells, including ols, neurons, and astrocytes. we demonstrate here that primary embryonic cortical tissue is an abundant cell source for both neurons and opcs that are myelination competent, easier to culture than rgcs, and widely used in large-scale hts screening within the pharmaceutical industry. we have validated this assay using gsis, establishing ec values for four different compounds to allow the ranking of potency. using this assay as an initial screen, we screened the ncc library and identified ten confirmed hit compounds from diverse target classes for follow-up characterization. perhaps the most important aspects of the cortical myelination assay are that ols develop and differentiate alongside growing axons and astrocytes, two major sources of signaling molecules known to influence myelination. the expression of the axonal protein lrr and ig domain-containing, nogo receptor-interacting protein was demonstrated be a potent inhibitor of differentiation and myelination. indeed, anti-lingo- antibody is being developed as an ms therapeutic to promote axon remyelination and is currently in human clinical trials. leukemia inhibitory factor has been shown to be released by astrocytes in response to atp from action potential firing axons to promote myelination. additionally, through the action of tnfr on astrocytes, lif is produced to stimulate ol differentiation in a co-culture system. furthermore, astrocytes were demonstrated to reduce ol differentiation, but specifically enhance myelin thickness and the rate of axon wrapping. tnf impairs ol differentiation attenuating tnf signaling by tnfr blocking therapy ameliorates ms symptoms in eae. it was also demonstrated that inhibiting glial γ-secretase promoted myelination, which also showed efficacy in vivo in the eae model of ms. these observations emphasize the importance of having culture conditions that more closely mimic the in vivo cns composition. it is important to note that this cortical myelination assay may fail to identify all possible compounds that may promote myelination. due to the relatively short time course and single compound addition, our assay may not identify compounds with short half-lives, require multiple target activations, or require longer time courses. the assay could be modified by spiking in test compounds during the course of the ensheathment window and/or lengthen the ensheathment window longer than 8 days. we included t forskolin, and cntf in the mym medium as factors that facilitate ol differentiation and survival. the activity of these factors may mask effects of potential stimulators of myelination. in particular, elimination of t may lower the threshold for identifying additional candidate compounds. this stimulation of differentiation by t may account for the lack of ol differentiation activity of benztropine and clemastine in our cortical myelination assay. elimination of these factors from the myelination phase of the assay may reveal additional compounds with myelination activity. the cortical myelination assay identifies compounds not revealed by ol differentiation assays our myelination assay greatly differs from in vitro ol differentiation assays used for compound screening which have only assessed differentiation using purified opcs adapted to culture conditions by multiple passages, or differentiated from induced pluripotent stem cells and carried out in very short developmental time frames. mei et al, developed an hts assay incorporating ol differentiation in the presence of inert micropillers allowing the quantification of pillar wrapping as a surrogate for myelination. lead compounds identified from these three studies, including clemastine, benztropine, miconazole and clobetasol, facilitate ol differentiation in cultures of purified opcs, but had no effect on myelination in our live axon myelination assay. notably, our myelination assay did not identify muscarinic antagonists as previously identified by independent ol differentiation screens, but revealed entirely new classes of compounds that promote myelination. no other high throughput assay has been developed to date capable of assessing the initiation and facilitation of myelination in the presence of axons, arguably the most important features when selecting candidate compounds capable of promoting or generating remyelination. relation of cortical myelination assay hit compounds to clinical applications and multiple sclerosis repositioning approved drugs for the treatment of new indications is an activity that has grown in popularity in recent years and is a trend that is predicted to continue. there are many examples where repurposed drugs are making an impact in the field of ms. a few to note are bg-, a modification of fumaderm®, used to treat psoriasis; fingolimod, originally developed for transplantation and alemtuzumab, an oncology drug. we found that eight out of our ten confirmed hit compounds aligned with current ms repositioning efforts and are briefly cited here. a treatment for congestive heart failure, digoxin enhances both myelination and differentiation in our assay. it has been shown to reverse conduction block in demyelinated nerve fibers in experimental animals. moreover, digoxin and possibly its derivatives, have also been implicated as a possible therapeutic for ms. a clinical study involving ms patients, showed improvement of symptoms when given the drug intravenously. other hits have shown efficacy in ms animal models. imatinib mesylate, a kinase inhibitor used for cancer treatment, is efficacious in experimental autoimmune encephalomyelitis models. artesunate, a natural compound used to treat malaria, has a derivative form that has anti-inflammatory properties in eae. methotrexate, an anti-metabolite used to treat rheumatoid arthritis, is also efficacious at restoring myelin in cuprizone-induced demyelination mouse models. in addition, methotrexate is commonly used off-label in ms, and is suggested as a low-dose, add-on therapy in combination with other treatments, to reduce the relapsing-remitting rate in ms and to slow the progression of ms. some hits have been evaluated or will soon be tested in human ms patients. oxcarbazepine, a treatment for seizures and amilioride, a treatment for high blood pressure, have been demonstrated to be neuroprotective in rodent models of ms and in ms patients, and are now in human clinical trials to treat progressive ms. additionally, there is a patent for docetaxel, an anti-microtubule and treatment for cancer on its use to treat or prevent psoriasis and ms. off-label muscle relaxants are used, when necessary, to help ms patients offset or reduce relapsing-remitting episodes. atracurium besylate is used as an adjunct to general anesthesia and particularly useful for ms patients undergoing surgery since general anesthesia can exacerbate ms symptoms. in a clinical case report, a patient with ms underwent surgery for renal failure, and in a 3 month follow-up, reported no further ms attacks. the remainder of our confirmed hits, zu-capsaicin and tegafur have not been tested in any demyelinating diseases. to our knowledge the myelin restorative effects of these compounds have not been evaluated. our data suggests that these drugs should additionally focus on testing these hits to determine whether they have beneficial effects on remyelination in vivo and possibly in ms patients.  we have developed the first high capacity, phenotypic in vitro assay amenable to high throughput screening to identify compounds that promote myelination. our myelination assay incorporates both live axons and astrocytes, both of which have profound effects on myelination in vivo. our assay utilizes primary cortical cells under conditions that allows for endogenous myelination, and thus avoids potential artefacts due to the use of cell lines or artificial substrates for myelination. to demonstrate the utility of this assay, we have screened the ncc library of fda-approved compounds, identifying ten compounds for follow up studies, including effects on different cell types, mechanisms of action, structure–activity relationship determination, and in in vivo models of ms. our hope is that this cortical myelination assay will be scaled up to screen even larger compound libraries in search of more efficacious ms therapeutics. 
we established a high throughput cell-based assay to identify compounds that promote myelination. myelinationoligodendrocytehigh throughputdrug screeningdifferentiationprimary cell-based assayimage analysismultiple sclerosismyelin basic proteinissue-copyright-statement© the author 2016 identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for ms patients. we have designed the first high capacity in vitro assay that assesses myelination of live axons. we have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. we have validated the assay scalability and consistency by screening the nih clinical collection library of compounds and identified ten compounds that promote myelination.
 regeneration of new myelin is impaired in persistent multiple sclerosis lesions, leaving neurons unable to function properly and subject to further degeneration. current ms therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system. development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid cns culture system.  we established a high throughput cell-based assay to identify compounds that promote myelination. culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. we developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a well plate format. we have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. we have validated the assay scalability and consistency by screening the nih clinical collection library of compounds and identified ten compounds that promote myelination. half maximal effective concentration values for these compounds were determined to rank them according to potency.  we have designed the first high capacity in vitro assay that assesses myelination of live axons. this assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for ms patients. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords myelinationoligodendrocytehigh throughputdrug screeningdifferentiationprimary cell-based assayimage analysismultiple sclerosismyelin basic proteinissue-copyright-statement© the author 2016 