Lysolecithin induces demyelination in vitro in a cerebellar slice culture system

Abstract

Demyelination is a hallmark of several human diseases, including multiple sclerosis. To understand better the process of demyelination and remyelination, we explored the use of an in vitro organotypic cerebellar slice culture system. Parasagittal slices of postnatal Day 10 (P10) rat cerebella cultured in vitro demonstrated significant myelination after 1 week in culture. Treatment of the cultures at 7 days in vitro (DIV) with the bioactive lipid lysolecithin (lysophosphatidylcholine) for 15-17 hr in vitro produced marked demyelination. This demyelination was observed by immunostaining for the myelin components myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). After a transient demyelinating insult with lysolecithin in vitro, the cultures recovered with oligodendrocyte differentiation recapitulating a normal time course; there was initially re-expression of CNPase and MBP during this recovery, and this was followed by MOG. In addition, there seemed to be some limited remyelination during the recovery phase. Lysolecithin thus induces demyelination in an in vitro organotypic cerebellar slice culture system, providing a model system for studying myelination, demyelination, and remyelination in vitro.

Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons

“…Myelinating cerebellar organotypic slice cultures from P8-9 WT and syntaphilin-KO mice were generated, and Purkinje cells were infected with lentiviral constructs containing mitochondria-targeted DsRed2 as reported previously (18). To induce demyelination, slices were incubated with media containing 0.05% wt/vol lysolecithin (L-α-lysophosphatidylcholine; Sigma-Aldrich) for 26 h and maintained without lysolecithin for ∼24 h before time-lapse imaging and/or fixation (51). The effect of 0.05% lysolecithin to axons is largely attributable to demyelination, as there is no significant effect on unmyelinated axons (17).…”

Multiplexed Dendritic Targeting of α Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway

“…Microtome sections were cut at 50 m and stored in PBS until staining at 4°C. Staining was performed as described previously, with slight modifications (Birgbauer et al, 2004). In brief, floating sections were incubated with 0.3% hydrogen peroxide in PBS for 30 min followed by blocking for 2-3 h in blocking solution (1% bovine serum albumin [BSA], 10% horse serum, and 0.2% Triton X-100 in PBS) at RT.…”

Mechanisms Involved in the Remyelinating Effect of Sildenafil

“…On the other hand, cerebellar organotypic cultures demyelinated with lysophosphatidylcholine (LPC) have proven to be an excellent model to study demyelination-remyelination process without axonal death and excluding the influence of the immune system. This model is accessible to experimental perturbation, which allows testing the effect of different stimuli on the mechanisms that might be implied in the processes that 83 wants to be studied (Birgbauer et al 2004, Zhang H. et al 2011. After LPC demyelination, microglia activation can be observed, myelin debris is generated as a consequence of demyelination and naked axons sometimes suffer damage which is evident by the presence of axonal bulbs, Caspr proteins are no longer visible, OPCs are present in the slices and after demyelination…”

“…Medium was changed every 2-3 days. After 7 days in vitro (DIV), the slices were demyelinated using L-α-lysophosphatidylcholine (LPC) from egg yolk (0.5 mg/ml) for 14 h. After that time medium was replaced with LPC-free medium (Birgbauer et al 2004). Treatments with specific activators or inhibitors of cGMP mediated pathways and kinases (Fig 2) were started at 9 DIV (Fig 1), and medium were replaced every 2-3 days until 19 DIV.…”

“…Cultures were established from cerebella of 7-day-old C57BL/6 mice as described in Methods (section 3.1). After 7 days in vitro (DIV), cerebellar slices were exposed to LPC (0.5 mg/ml) for 14-17h, a treatment that causes a dramatic demyelination without axonal or cell death (Birgbauer et al 2004). Twenty-four hours after removing LPC, cultures were treated with four doses of sildenafil (1 µM shows that the remyelination induced by sildenafil alone is similar to that attained in combination with BAY41 (BAY41+Sil) or NMDA (NMDA+Sil) (Fig 2B).…”