Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis

Abstract

Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as 'rings' of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis.

Figure 1

Conception and fabrication of micropillar arrays for modeling myelination. (a) An oligodendrocyte wrapping nanofibers, visualized by MBP immunostaining. Scale bar, 20 μm. (b) Compressed z-stack image of oligodendrocytes wrapping nanofibers, visualized from the side and detected as ‘rings’ of myelin membrane (arrows, MBP-positive staining). Scale bar, 10 μm. (c,d) Conical micropillars, fabricated from fused silica and patterned for bonding into 96-well bottomless plates, imaged by SEM; interpillar distances and the physical dimensions of the pillars are also shown. (e) Illustration of the concept for micropillar arrays as a binary indicant for membrane wrapping. Using an inverted confocal microscope, the pillars can be imaged from the bottom of the plate, and OPC and oligodendrocyte membrane wrapping can be visualized and quantified. (f) Schematic diagram of a hypothesized compressed z-stack two-dimensional image of an oligodendrocyte wrapping a micropillar. Conceptually, a single high-resolution fluorescence image will allow for the measurement of the tip diameter and base diameter of membrane wrapping, as well as the corresponding extent and length in the z-plane. (g) Low-field SEM image of oligodendroglia interacting with and wrapping micropillars. Arrows show multiple OPCs interacting with single pillars, and arrowheads show oligodendrocytes paired with a single pillar. Scale bar, 10 μm.

Figure 2

BIMA. (a) Oligodendroglial cells interacting with the micropillars, detected by fluorescence confocal microscopy as green (PDGFRα-positive OPCs) or red (MBP-positive oligodendrocyte) rings. Scale bar, 25 μm. (b) High-magnification image of a 10-μm compressed z-stack image of an oligodendrocyte and a neighboring OPC. Scale bar, 10 μm. (c) A single oligodendrocyte XView image and with a 30° tilt from the side view. (d) SEM image of a single oligodendrocyte wrapping a micropillar. The arrow indicates an oligodendrocyte cell body. Scale bar, 1 μm. (e) High-resolution SEM image of the corresponding inset box in d illustrating multiple concentric layers of membrane made by the oligodendrocyte (as indicated by the arrows). Scale bar, 1 μm.

Figure 3

High-throughput screening of bioactive compounds for differentiation and membrane wrapping identifies a cluster of antimuscarinic compounds. (a) Approximately 500 compounds were plotted based on the percentage of MBP-positive and PDGFRα-positive rings. Each compound was quantified and averaged based on four 100-micropillar fields from each array performed in triplicate. Based on the control arrays and known samples (PDGF, T3 and XAV939), compounds were categorized into four quadrants representing proliferation, apoptosis, differentiation and the combination of both proliferation and differentiation. The control (black diamond) is represented at the intersection of the green and red lines that indicate the average measurement of MBP-positive and PDGFRα-positive rings, as well as the extent of the s.e.m. associated with the control measurements. (b–e) Representative 100-micropillar fields immunostained for MBP (red) and PDGFRα (green) for control arrays (b), T3 (c), clemastine (d) and benzatropine (e). Scale bar, 100 μm. (f) Quantification for control, T3, clemastine, benzatropine, quetiapine, oxybutynin, trospium and ipratropium from the compound screen. Data are plotted as the number of MBP- or PDGFRα-positive rings in each field of 100 micropillars. Error bars represent mean ± s.e.m. *P < 0.05, significance based on Student’s t-test with the respective controls.

Figure 4

Validation of clemastine and benzatropine with purified oligodendroglia cultured alone or with purified DRG neurons. (a–d) Control cultures (a) or cultures treated with T3 (b), clemastine (c) or benzatropine (d) for 3 d immunostained for MBP (red) and PDGFRα (green). Scale bar, 100 μm. (f–i) Control cocultures (f) or cultures treated with T3 (g), clemastine (h) or benzatropine (i) immunostained for MBP (red), PDGFRα (green) and neurofilament (white). Each of the single color images is displayed in a merged format with all three colors. Cell nuclei are identified by DAPI (blue). Scale bar, 50 μm. (e,j) Quantification of the percentages of MBP- and PDGFRα- positive cells from the purified OPC cultures (e) or DRG cocultures (j) in the presence of T3, clemastine or benzatropine. Error bars represent mean ± s.e.m., and all experiments were performed in triplicate. *P < 0.05, significance based on Student’s t-test with the respective controls. n = 3 for all experiments.

Figure 5

Clemastine enhances the kinetics of remyelination and promotes remyelination in mice after gliotoxic injury with lysolecithin. (a) Dark-field micrograph of an adult mouse spinal cord illustrates focal demyelinated lesions in the dorsal funiculus and ventrolateral white matter. Scale bar, 300 μm. (b) Focal demyelinated lesions induced by injection of lysolecithin (n = 6) in mice treated with or without clemastine. In situ hybridization of plp in the lesions were examined after oral administration of clemastine at 7 and 14 days post lesion (d.p.l.). Scale bar, 100 μm. (c,d) Mice at 14 d.p.l. were subjected to Cnp1 in situ hybridization (c) and MBP staining (d) following the administration of clemastine and demyelination. Dashed lines demarcate lesion areas. Scale bar in b applies to c and d. (e) Quantification of plp in situ hybridization. Error bars represent mean ± s.d., and all experiments were performed in quadruplicate. *P = 0.05, **P = 0.009, significance based on Student’s t-test. (f) Quantification of myelin sheath thickness and the proportion of myelinated and unmyelinated axons in control (blue) and clemastine-treated (red) mice at d.p.l. by g-ratio analysis. The scatterplot displays g-ratios of individual axons as a function of axonal diameter. All g-ratios were analyzed from transmission electron microscopy images. (g,h) Representative electron microscopy images for control (g) and clemastine-treated (h) mice at 14 d.p.l. Scale bar, 2 μm.