Investigating demyelination, efficient remyelination and remyelination failure in organotypic cerebellar slice cultures:Workflow and practical tips

Healthy myelin is essential for proper brain function. When the myelin sheath is damaged, fast saltatory impulse conduction is lost and neuronal axons become vulnerable to degeneration. Thus, regeneration of the myelin sheath by encouraging oligodendrocyte lineage cells to remyelinate the denuded axons is a promising therapeutic target for demyelinating diseases such as multiple sclerosis. Ex vivo organotypic cerebellar slice cultures are a useful model to study developmental myelination, demyelination, remyelination and remyelination failure. In these cultures, the cerebellum's three-dimensional architecture and various cell populations remain largely intact, providing a realistic and relatively cost-efficient model that can be easily manipulated by the addition of viral vectors, pharmaceuticals or (transgenic) cells to augment or replace resident cell populations. Moreover, slice cultures can be treated with lysolecithin or polyinosinic:polycytidylic acid to induce demyelination and mimic efficient as well as inefficient remyelination. It can be challenging to set up slice cultures for the first time, as in our experience, seemingly minor differences in technique and materials can make a great difference to the quality of the cultures. Therefore, this report provides an in-depth description for the generation and maintenance of ex vivo organotypic cerebellar cultures for demyelination-remyelination studies with a focus on practical tips for scientists that are new to this technique

The role of arachidonic acid metabolism in intestinal pathogenesis

Arachidonic acid (AA) metabolism is a critical process mediated by a number of enzymes that control physiological and pathological functions. The first study demonstrates that administration of COX-inhibitors to mice with a genetic deletion in cPLA2 causes acute lethality within two weeks, with no effect on wild-type littermates. Morbidity was a result of severe GI damage with associated bacteremia and sepsis. Further analysis revealed genes involved in ATP generation to be differentially expressed when comparing genotypes after drug administration. cPLA2-/- mice had morphological damage of enterocytic mitochondria with subsequent impairment of ATP generation. This study demonstrates a protective role for AA generation against drug-induced GI toxicity related to mitochondria] damage and impaired function. The second study examines the protective roles of PGs against ulcerative colitis (UC). Administration of dextran sodium sulfate (DSS) to mice with a genetic deletion in cPLA2 induced greater clinical and pathological disease as compared to wild-type littermates. A subsequent study demonstrated that DSS exposure to mice with a genetic deletion in mPGES-1 also suffered more extensive acute disease than wild-type littermates. mPGES-1-/- mice were also shown to be impaired in their ability to recover from DSS-induced injury related to expansion of colonic ulcerations. This study demonstrates a protective role for PGE2 against susceptibility to acute UC disease as well as recovery. The third study investigates the impact of the inflammatory response on intestinal tumorigenesis. Exposure of DSS to APCmin/+ mice with a genetic deletion of mPGES-1 decreases intestinal tumor number, with no effect in APCmin/+ mice. No changes in epithelial markers within tumors were observed in any group, but examination of tumor-associated myeloid derived suppressor cells showed impaired ARG-1 expression. This study demonstrates that DSS administration to a mouse model that has an altered inflammatory response results in decreased intestinal tumor development. Taken together, these three studies demonstrate the protective roles of components of the AA metabolism cascade in the GI tract.

cPLA2 Is Protective Against COX Inhibitor–Induced Intestinal Damage

Cytosolic phospholipase A2 (cPLA2) is the rate-limiting enzyme responsible for the generation of prostaglandins (PGs), which are bioactive lipids that play critical roles in maintaining gastrointestinal (GI) homeostasis. There has been a long-standing association between administration of cyclooxygenase (COX) inhibitors and GI toxicity. GI injury is thought to be induced by suppressed production of GI-protective PGs as well as direct injury to enterocytes. The present study sought to determine how pan-suppression of PG production via a genetic deletion of cPLA2 impacts the susceptibility to COX inhibitor–induced GI injury. A panel of COX inhibitors including celecoxib, rofecoxib, sulindac, and aspirin were administered via diet to cPLA2− / − and cPLA2+ / + littermates. Administration of celecoxib, rofecoxib, and sulindac, but not aspirin, resulted in acute lethality (within 2 weeks) in cPLA2− / − mice, but not in wild-type littermates. Histomorphological analysis revealed severe GI damage following celecoxib exposure associated with acute bacteremia and sepsis. Intestinal PG levels were reduced equivalently in both genotypes following celecoxib exposure, indicating that PG production was not likely responsible for the differential sensitivity. Gene expression profiling in the small intestines of mice identified drug-related changes among a panel of genes including those involved in mitochondrial function in cPLA2− / − mice. Further analysis of enterocytic mitochondria showed abnormal morphology as well as impaired ATP production in the intestines from celecoxib-exposed cPLA2− / − mice. Our data demonstrate that cPLA2 appears to be an important component in conferring protection against COX inhibitor–induced enteropathy, which may be mediated through affects on enterocytic mitochondria