Diazoxide promotes oligodendrocyte precursor cell proliferation and myelination

Abstract

Background: Several clinical conditions are associated with white matter injury, including periventricular white matter injury (PWMI), which is a form of brain injury sustained by preterm infants. It has been suggested that white matter injury in this condition is due to altered oligodendrocyte (OL) development or death, resulting in OL loss and hypomyelination. At present drugs are not available that stimulate OL proliferation and promote myelination. Evidence suggests that depolarizing stimuli reduces OL proliferation and differentiation, whereas agents that hyperpolarize OLs stimulate OL proliferation and differentiation. Considering that the drug diazoxide activates K(ATP) channels to hyperpolarize cells, we tested if this compound could influence OL proliferation and myelination.

Methodology/findings: Studies were performed using rat oligodendrocyte precursor cell (OPC) cultures, cerebellar slice cultures, and an in vivo model of PWMI in which newborn mice were exposed to chronic sublethal hypoxia (10% O(2)). We found that K(ATP) channel components Kir 6.1 and 6.2 and SUR2 were expressed in oligodendrocytes. Additionally, diazoxide potently stimulated OPC proliferation, as did other K(ATP) activators. Diazoxide also stimulated myelination in cerebellar slice cultures. We also found that diazoxide prevented hypomyelination and ventriculomegaly following chronic sublethal hypoxia.

Conclusions: These results identify KATP channel components in OLs and show that diazoxide can stimulate OL proliferation in vitro. Importantly we find that diazoxide can promote myelination in vivo and prevent hypoxia-induced PWMI.

Figure 1. PreOLs express KIR6.1, KIR6.2, SUR1 and SUR2 genes.

cDNA was prepared from OPCs and used in PCR reactions with primers specific to each gene. 98% of cells were A2B5-postive indicating that cells were OPCs. Left, molecular weight markers. White bands show amplified products. Data shown are representative of three separate studies performed on OPC cultures prepared at different times. Similar observations (not shown) were seen for mature OLs (95% MBP-positive).

Figure 2. OPCs express KIR6.1.

OLs derived from neonatal rat brain were isolated and cultured. Double-labeling immunostaining shows staining for KIR6.1 in either A2B5 or O1-positive OLs. Data shown are representative of three separate studies performed on OPC cultures prepared at different times.

Figure 3. OPCs express SUR and KIR protein.

OPCs derived from neonatal rat brain were isolated and cultured. Western blotting was performed on whole brain lysates and OPCs. Approximate sizes of bands were SUR1, 140 kDa; SUR2, 180 kDa; Kir6.1 70 kDa; Kir6.2, 60 kDa. OL, oligodendrocyte cultures; Br, whole brain. Data shown are representations of three separate studies using OPCs prepared at different times.

Figure 4. Concentration-response effects of diazoxide on PreOL proliferation.

Data shown are from three studies in which each concentration was tested in triplicate in each study. * p<0.05, vs. vehicle, ANOVA. Mean ± SEM shown. Y-axis represents the percent increase relative to control in cellular DNA content, which is a direct index of cell proliferation.

Figure 5. Effects of known K_ATP channel activators on OPC proliferation.

Diazoxide (10 uM), ZM26600 (2.5 uM), Pinacidil (10 uM), Y26763 (200 nM), Levcromakalim (2.5 uM), P1075 (100 nM). n = .5-10 (from two separate experiments). Data shown are from three studies in which each concentration was tested in triplicate in each study. * p< 0.05, vs. vehicle, ANOVA. Mean ± SEM shown. Y-axis represents the percent increase relative to control in cellular DNA content, which is a direct index of cell proliferation.

Figure 6. Diazoxide inhibits intracellular calcium accumulation.

Data points are the mean ± SEM of 30 separate cells. 100 depicts baseline Fluo-3 intensity. Drugs administered at 0 seconds. These data are representative of 3 separate studies. Tolbutamide (100 uM), diazoxide (10 uM).

Figure 7. Diazoxide stimulates myelinated fiber formation.

Slice cultures from P0 cerebellum were treated with diazoxide (1 uM), tolbutamide (100 uM), or vehicle for 5 days. Slices were stained for MBP. A. Top panel shows quantitative assessment of myelinated fiber number (p<0.01; ANOVA). Data are mean ± SEM from six separate slices per treatment. B. Images of MBP labeled specimens.

Figure 8. Animals reared in chronic hypoxia and treated with diazoxide demonstrate reductions in ventriculomegaly.

A. Top panel shows quantitative assessment of ventricle size. Data shown are mean ± SEM from one experiment with 4–6 animals per treatment group (p<0.01; t-test). Similar results were obtained in another separate study performed at a different time. Chronic hypoxia caused pronounced ventriculomegaly (arrow). Note the reduction in ventricle size in the mice treated with diazoxide. B. Photographs are from one animal in each treatment group. * p<0.01 ANOVA.

Figure 9. Animals reared in chronic hypoxia and room air demonstrate increased myelination with diazoxide treatment.

Coronal images at level of corpus callosum shown are from one experiment with 4–6 animals per treatment group and are representative of one other separate study performed at a different time. MBP staining was performed at the same time. Box depict region of corpus callosum where labeling intensity was assessed. Photographs were taken at identical exposures. Hypoxia caused diffuse reduction in cerebral MBP-labeling, which was markedly improved with diazoxide. We also observed more MBP-labeling in diazoxide-treatment mice reared in room air compared to those treated with vehicle.

Figure 10. Animals reared in chronic hypoxia and room air demonstrate increased myelination with diazoxide treatment.

Quantitative assessment of labeling using Image J Version 1.42q (National Institutes of Health, Bethesda MD) at the mid-level of the corpus callosum of groups shown in Fig. 9. Data shown are mean ± SEM from one experiment with 3–6 animals per treatment group. Similar results were obtained in another separate study performed at a different time.