Organotypic Cerebellar Cultures: 300µm Parasagittal Slices of Mouse Cerebellum cut with 5100mz

Abstract

(from https://doi.org/10.1007/s11481-021-09991-6)

Oligodendrocytes produce the myelin that is critical for rapid neuronal transmission in the central nervous system (CNS).

Disruption of myelin has devastating effects on CNS function, as in the demyelinating disease multiple sclerosis (MS).

Microglia are the endogenous immune cells of the CNS and play a central role in demyelination and repair. There is a need for new potential therapies that regulate myelination and microglia to promote repair. Agathisflavone (FAB) is a non-toxic flavonoid that is known for its anti-inflammatory and neuroprotective properties. Here, we examined the effects of FAB (5-50μM) on myelination and microglia in organotypic cerebellar slices prepared from P10-P12 Sox10-EGFP and Plp1-DsRed transgenic mice. Immunofluorescence labeling for myelin basic protein (MBP) and neurofilament (NF) demonstrates that FAB significantly increased the proportion of MBP + /NF + axons but did not affect the overall number of oligodendroglia or axons, or the expression of oligodendroglial proteins CNPase and MBP. FAB is known to be a phytoestrogen, but blockade of α- or β- estrogen receptors (ER) indicated the myelination promoting effects of FAB were not mediated by ER. Examination of microglial responses by Iba1 immunohistochemistry demonstrated that FAB markedly altered microglial morphology, characterized by smaller somata and reduced branching of their processes, consistent with a decreased state of activation, and increased Iba1 protein expression. The results provide evidence that FAB increases the extent of axonal coverage by MBP immunopositive oligodendroglial processes and has a modulatory effect upon microglial cells, which are important therapeutic strategies in multiple neuropathologies.

Method

The effects of FAB (Agathisflavone) were examined ex vivo in the organotypic cerebellar slice model of myelination (Doussau et al. 2017), using P10-12 mice, according to the methods previously described (Stoppini et al. 1991; Simoni and Yu 2006). In brief, 300 μm parasagittal slices of the cerebellum were cut using a 5100 mz vibrating microtome (Campden Instruments LTD). Four to six cerebellar slices were transferred to each half-millicell membrane insert (Millipore, 30 mm

diameter, pore size 0.4 μm) and cultured using the interface method (Stoppini et al. 1991), with 1 mL of serum-based

medium, composed of 50% Minimum Essential Medium with Glutamax-1, 18% Earle’s Balanced Salt Solution (EBSS),

5% EBSS + D-glucose, 1% penicillin–streptomycin, and 10% horse serum (Gibco Invitrogen; (Simoni and Yu 2006)).

Slices were maintained in media for 3 days in vitro (3DIV) at 37ºC, under standard conditions (95% O2, 5% CO2).