(A–C) Rat slices cultured 21 days display extensive myelination. (A) Nodes of Ranvier identified with CASPR (red arrowheads). (B) Representative ultrastructural images of early (left panel) and late (right panel) myelination and detail of multilamellar myelin sheaths (insets). (C) Schematic of strategy to analyze chronic in vitro myelination. prep, prepared from. Representative images of myelinated axons in callosal WM visualized with MBP and 200 kDa neurofilament H (NFH) after treatment with DMSO (vehicle), MDa HA (50 nM) with or without VCPAL (25 μM), or bHAf (100 nM). (D) 175–300 kDa HAf regulates OPC maturation cell autonomously. Primary OPCs cultured 4 days under pro-differentiation conditions stained for OPCs (PDGFRα, green), OLs (MBP, red), and DAPI (blue). Cultures treated with PBS (vehicle), MDa HA (50 nM), HAf 175–300 kDa (500 nM), or HAf 5–20 kDa (500 nM). Quantification of total OPCs and OLs. (E) MBP⁺ OLs in DIV8 slices cultured with PBS (vehicle) or HAf of 40 (100 nM), 106 (100 nM), 210 (100 nM) or 357 kDa (100 nM). (F) Primary OPCs differentiated 4 days with or without bHAf (500 nM) visualized with cyclic nucleotide phosphodiesterase (CNP), MBP, and DAPI (left). bHAF treatment decreased the percentage of OLs (right). (G) Density of OLs in DIV8 slices cultured with varying bHAf concentrations. (H) OL quantification in DIV8 slices cultured with bHAf (100 nM) and equimolar concentrations of HAf at 357 or 106 kDa or combined with 357-kDa and 106-kDa HAf (100 nM). A, C, and E: n = 3 animals/condition from separate litters; 3 slices/treatment condition/animal, 9 slices total. D and F: n = 2 separate culture preparations. G and H: n = 1 animal per condition; 3 slices analyzed per treatment condition. *P < 0.05 by Student’s t test; **P < 0.001 by ANOVA; mean ± SD. Scale bars: 12 μm, A; 600 nm, B (insets: original magnification, ×33,000); 150 μm, C; 75 μm, D and F.