Evaluation of treatment of experimentally induced canine model of multiple sclerosis using laser activated non-expanded adipose derived stem cells.

Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system that destroys oligodendrocytes. This work aims to evaluate the treatment of experimentally induced MS in dogs using laser activated non-expanded adipose derived stem cells. The results showed amelioration of the clinical signs over time confirmed by the resolution of the previous lesions on MRI. Positive migration of the injected cells to the site of lesion, increased remyelination detected by Myelin Basic Proteins, positive differentiation into Olig2 positive oligodendrocytes, prevented the glial scar formation and restored axonal architecture. The study concluded that treatment using laser activated stem cells holds a promising therapeutic option for treatment of MS in a canine model.

Age-Dependent Responses of Brain Myelin Integrity and Behavioral Performance to Radiation in Mice.

Radiation therapy is widely used to treat primary and metastatic brain tumors, but it may also lead to delayed neurological complications. Oligodendrocytes in the central nervous system produce myelin, and myelin integrity becomes highly vulnerable after brain irradiation. In this study, mice at different developmental stages were used to test the age-dependent sensitivity of myelin formation and maintenance, as well as behavioral performance after whole-brain irradiation (WBI). Mice at postnatal days 21 and 28 and at 2 months received a single dose of 25 Gy WBI. Behavioral tests for general locomotor activity and motor coordination revealed an age-dependent response after WBI. Quantitative observation revealed a sharp decrease in the number of oligodendrocytes beginning at day 1 after WBI, which recovered during different observation intervals in white matter and gray matter in mice of different ages. Myelin basic protein (MBP) staining revealed disparate quantities in an age- and brain-region-dependent pattern between groups after WBI, which was confirmed using Black-Gold staining. In summary, the response to radiation in mice of different ages provided insight into the potential of oligogenesis in microenvironments at respective stages of myelin regeneration, which may reduce central nervous system impairment and optimize the prognosis after radiation treatment.

Fractionation Spares Mice From Radiation-Induced Reductions in Weight Gain But Does Not Prevent Late Oligodendrocyte Lineage Side Effects.

PURPOSE: To determine the late effects of fractionated versus single-dose cranial radiation on murine white matter. METHODS AND MATERIALS: Mice were exposed to 0 Gy, 6 × 6 Gy, or 1 × 20 Gy cranial irradiation at 10 to 12 weeks of age. Endpoints were assessed through 18 months from exposure using immunohistochemistry, electron microscopy, and electrophysiology. RESULTS: Weight gain was temporarily reduced after irradiation; greater loss was seen after single versus fractionated doses. Oligodendrocyte progenitor cells were reduced early and late after both single and fractionated irradiation. Both protocols also increased myelin g-ratio, reduced the number of nodes of Ranvier, and promoted a shift in the proportion of small, unmyelinated versus large, myelinated axon fibers. CONCLUSIONS: Fractionation does not adequately spare normal white matter from late radiation side effects.

Delayed brain radiation necrosis: pathological review and new molecular targets for treatment.

Delayed radiation necrosis is a well-known adverse event following radiotherapy for brain diseases and has been studied since the 1930s. The primary pathogenesis is thought to be the direct damage to endothelial and glial cells, particularly oligodendrocytes, which causes vascular hyalinization and demyelination. This primary pathology leads to tissue inflammation and ischemia, inducing various tissue protective responses including angiogenesis. Macrophages and lymphocytes then infiltrate the surrounding areas of necrosis, releasing inflammatory cytokines such as interleukin (IL)-1α, IL-6, and tumor necrosis factor (TNF)-α. Microglia also express these inflammatory cytokines. Reactive astrocytes play an important role in angiogenesis, expressing vascular endothelial growth factor (VEGF). Some chemokine networks, like the CXCL12/CXCR4 axis, are upregulated by tissue inflammation. Hypoxia may mediate the cell-cell interactions among reactive astrocytes, macrophages, and microglial cells around the necrotic core. Recently, bevacizumab, an anti-VEGF antibody, has demonstrated promising results as an alternative treatment for radiation necrosis. The importance of VEGF in the pathophysiology of brain radiation necrosis is being recognized. The discovery of new molecular targets could facilitate novel treatments for radiation necrosis. This literature review will focus on recent work characterizing delayed radiation necrosis in the brain.

Histone H3F3A and HIST1H3B K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes.

Diffuse intrinsic pontine glioma (DIPG) is the most severe paediatric solid tumour, with no significant therapeutic progress made in the past 50 years. Recent studies suggest that diffuse midline glioma, H3-K27M mutant, may comprise more than one biological entity. The aim of the study was to determine the clinical and biological variables that most impact their prognosis. Ninety-one patients with classically defined DIPG underwent a systematic stereotactic biopsy and were included in this observational retrospective study. Histone H3 genes mutations were assessed by immunochemistry and direct sequencing, whilst global gene expression profiling and chromosomal imbalances were determined by microarrays. A full description of the MRI findings at diagnosis and at relapse was integrated with the molecular profiling data and clinical outcome. All DIPG but one were found to harbour either a somatic H3-K27M mutation and/or loss of H3K27 trimethylation. We also discovered a novel K27M mutation in HIST2H3C, and a lysine-to-isoleucine substitution (K27I) in H3F3A, also creating a loss of trimethylation. Patients with tumours harbouring a K27M mutation in H3.3 (H3F3A) did not respond clinically to radiotherapy as well, relapsed significantly earlier and exhibited more metastatic recurrences than those in H3.1 (HIST1H3B/C). H3.3-K27M-mutated DIPG have a proneural/oligodendroglial phenotype and a pro-metastatic gene expression signature with PDGFRA activation, while H3.1-K27M-mutated tumours exhibit a mesenchymal/astrocytic phenotype and a pro-angiogenic/hypoxic signature supported by expression profiling and radiological findings. H3K27 alterations appear as the founding event in DIPG and the mutations in the two main histone H3 variants drive two distinct oncogenic programmes with potential specific therapeutic targets.

Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal.

Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to (137)Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure.

Regionally distinct responses of microglia and glial progenitor cells to whole brain irradiation in adult and aging rats.

Radiation therapy has proven efficacy for treating brain tumors and metastases. Higher doses and larger treatment fields increase the probability of eliminating neoplasms and preventing reoccurrence, but dose and field are limited by damage to normal tissues. Normal tissue injury is greatest during development and in populations of proliferating cells but also occurs in adults and older individuals and in non-proliferative cell populations. To better understand radiation-induced normal tissue injury and how it may be affected by aging, we exposed young adult, middle-aged, and old rats to 10 Gy of whole brain irradiation and assessed in gray- and white matter the responses of microglia, the primary cellular mediators of radiation-induced neuroinflammation, and oligodendrocyte precursor cells, the largest population of proliferating cells in the adult brain. We found that aging and/or irradiation caused only a few microglia to transition to the classically "activated" phenotype, e.g., enlarged cell body, few processes, and markers of phagocytosis, that is seen following more damaging neural insults. Microglial changes in response to aging and irradiation were relatively modest and three markers of reactivity - morphology, proliferation, and expression of the lysosomal marker CD68- were regulated largely independently within individual cells. Proliferation of oligodendrocyte precursors did not appear to be altered during normal aging but increased following irradiation. The impacts of irradiation and aging on both microglia and oligodendrocyte precursors were heterogeneous between white- and gray matter and among regions of gray matter, indicating that there are regional regulators of the neural response to brain irradiation. By several measures, the CA3 region of the hippocampus appeared to be differentially sensitive to effects of aging and irradiation. The changes assessed here likely contribute to injury following inflammatory challenges like brain irradiation and represent important end-points for analysis in studies of therapeutic strategies to protect patients from neural dysfunction.

Downregulation of PRDX1 by promoter hypermethylation is frequent in 1p/19q-deleted oligodendroglial tumours and increases radio- and chemosensitivity of Hs683 glioma cells in vitro.

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and linked to radio- and chemotherapy response as well as longer survival. The molecular mechanisms underlying this clinically important association are as yet unknown. Here, we studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio- and chemosensitivity of glioma cells in vitro. In total, we screened primary glioma tissues from 93 patients for methylation of the 5'-CpG island of PRDX1 by sodium bisulfite sequencing. PRDX1 mRNA and protein expression levels were determined in subsets of the tumours by quantitative PCR and western blot analysis, respectively. PRDX1 hypermethylation and reduced expression were frequently detected in oligodendroglial tumours and secondary glioblastomas, but not in primary glioblastomas. In oligodendroglial tumours, both PRDX1 hypermethylation and reduced mRNA expression were significantly associated with 1p/19q-deletion. Stable knockdown of PRDX1 by lentiviral transduction of short-hairpin (sh)RNA constructs significantly increased apoptosis and reduced cell viability of Hs683 glioma cells exposed to ionizing irradiation or temozolomide in vitro. Taken together, our findings indicate that epigenetic silencing of PRDX1 is frequent in 1p/19q-deleted oligodendroglial tumours and likely contributes to radio- and chemosensitivity of these tumours.

Maintenance of white matter integrity in a rat model of radiation-induced cognitive impairment.

Radiation therapy is used widely to treat primary and metastatic brain tumors, but also can lead to delayed neurological complications. Since maintenance of myelin integrity is important for cognitive function, the present study used a rat model that demonstrates spatial learning and memory impairment 12 months following fractionated whole-brain irradiation (WBI) at middle age to investigate WBI-induced myelin changes. In this model, 12-month Fischer 344 x Brown Norway rats received 9 fractions of 5 Gy delivered over 4.5 weeks (WBI rats); Sham-IR rats received anesthesia only. Twelve months later, the brains were collected and measures of white matter integrity were quantified. Qualitative observation did not reveal white matter necrosis one year post-WBI. In addition, the size of major forebrain commissures, the number of oligodendrocytes, the size and number of myelinated axons, and the thickness of myelin sheaths did not differ between the two groups. In summary, both the gross morphology and the structural integrity of myelin were preserved one year following fractionated WBI in a rodent model of radiation-induced cognitive impairment. Imaging studies with advanced techniques including diffusion tensor imaging may be required to elucidate the neurobiological changes associated with the cognitive impairment in this model.

Long-term impact of radiation on the stem cell and oligodendrocyte precursors in the brain.

BACKGROUND: The cellular basis of long term radiation damage in the brain is not fully understood. METHODS AND FINDINGS: We administered a dose of 25Gy to adult rat brains while shielding the olfactory bulbs. Quantitative analyses were serially performed on different brain regions over 15 months. Our data reveal an immediate and permanent suppression of SVZ proliferation and neurogenesis. The olfactory bulb demonstrates a transient but remarkable SVZ-independent ability for compensation and maintenance of the calretinin interneuron population. The oligodendrocyte compartment exhibits a complex pattern of limited proliferation of NG2 progenitors but steady loss of the oligodendroglial antigen O4. As of nine months post radiation, diffuse demyelination starts in all irradiated brains. Counts of capillary segments and length demonstrate significant loss one day post radiation but swift and persistent recovery of the vasculature up to 15 months post XRT. MRI imaging confirms loss of volume of the corpus callosum and early signs of demyelination at 12 months. Ultrastructural analysis demonstrates progressive degradation of myelin sheaths with axonal preservation. Areas of focal necrosis appear beyond 15 months and are preceded by widespread demyelination. Human white matter specimens obtained post-radiation confirm early loss of oligodendrocyte progenitors and delayed onset of myelin sheath fragmentation with preserved capillaries. CONCLUSIONS: This study demonstrates that long term radiation injury is associated with irreversible damage to the neural stem cell compartment in the rodent SVZ and loss of oligodendrocyte precursor cells in both rodent and human brain. Delayed onset demyelination precedes focal necrosis and is likely due to the loss of oligodendrocyte precursors and the inability of the stem cell compartment to compensate for this loss.

A different regional response by mouse oligodendrocyte progenitor cells (OPCs) to high-dose X-irradiation has consequences for repopulating OPC-depleted normal tissue.

This study was designed to investigate whether the residual, dysfunctional oligodendrocyte progenitor cells (OPCs) observed following X-irradiation of the mouse spinal cord [D. M. Chari et al. (2003) Exp. Neurol., 198, 145-153], the presence of which prevented the endogenous repopulation of these areas from normal tissue, reflects a general response of OPCs in the mouse central nervous system (CNS) to X-irradiation. The brains of adult mice were exposed to 40 Gy of X-irradiation and the effect of X-irradiation on the OPCs was assessed up to 4 weeks post-irradiation using anti-NG2 antibodies. X-irradiation resulted in almost complete depletion of OPCs within the telencephalon (cortex, corpus callosum and hippocampus) by 7 days post-irradiation, which was followed by progressive repopulation of OPCs from non-irradiated areas of the cortex. By contrast, within the lower brain centres (the diencephalon and mesencephalon) OPC loss occurred much more slowly so that 26% of the OPCs still remained 4 weeks after X-irradiation. The consequence of this heterogeneous response to X-irradiation was that whereas transplanted and endogenous OPCs rapidly established themselves in the OPC-depleted telencephalon this did not occur in the areas where there was incomplete depletion of endogenous OPCs. Our findings confirm not only the requirement for almost complete OPC depletion in order to establish transplanted OPCs in normal tissue but also highlight a heterogeneity of progenitor populations in different areas of the mouse CNS.

Crayfish mechanoreceptor neuron prevents photoinduced apoptosis of satellite glial cells.

Interactions between neurons and glia play a key role in the development, functioning and survival of the nervous system. However, the influence of neurons on glial cells has received less attention than the role of glia in supporting neural functions. We here investigated the role of isolated crayfish stretch receptor neuron in the death of satellite glial cells under photodynamic impact. After staining with aluminum phthalocyanine photosens, the neuronal cell body was locally irradiated with a focused beam of He-Ne (633 nm, 200 W/cm2) or semiconductor laser (650 nm, 50 W/cm2). This rapidly abolished neuronal activity. The whole preparation was then subjected to total laser irradiation with lower intensity (633 nm, 0.3 W/cm2), which induced death of glial cells. Double staining of the preparation with propidium iodide and Hoechst 33342 in the following 6-7h allowed the visualization of necrotic, apoptotic and alive cells. Previous neuron inactivation with the focused laser beam was found to increase photodynamically-induced apoptosis but not necrosis of satellite glial cells enwrapping the axon. Therefore, the intact neuronal cell body protected satellite glial cells against photoinduced apoptosis. Altogether the data indicate that mechanoreceptor neurons release some signaling molecules involved in the prevention of glial apoptosis. This may provide integrity of the stretch receptor organ and its resistance to injurious factors.

Repopulation of oligodendrocyte progenitor cell-depleted tissue in a model of chronic demyelination.

Some, but not all, chronically demyelinated multiple sclerosis (MS) lesions are depleted of oligodendrocyte progenitor cells (OPCs) suggesting that OPCs are destroyed during the process of demyelination and some factor impedes OPC repopulation of the depleted tissue. The chronically demyelinated axons in MS lie in an astrocytic environment and it has been proposed that this might impede entry of OPCs into such regions. By depleting a short length of spinal cord of its OPCs using 40 Gy of X-irradiation in both normal rats and rats with progressive myelin loss accompanied by an astrocytosis (taiep rats), we investigated whether such changes affect the ability of OPCs to repopulate OPC-depleted tissue. In both taiep and normal rats, the rate of repopulation decreases with age, but no difference was detected in the rate at which OPCs repopulated normally myelinated and chronically demyelinated and astrocytosed tissue. This indicates that, if the astrocytic environment of the taiep central nervous system (CNS) is comparable to that found in MS lesions, then the presence of chronically demyelinated axons and astrocytosis in chronic MS lesions does not represent a barrier to repopulation of the tissue by OPCs. However, similar to the situation in the normal adult rodent CNS, the rate of repopulation by endogenous OPCs in aged taiep rats is very slow, approximately 0.2 mm per week.

Radiation-induced apoptosis of oligodendrocytes in the adult rat optic chiasm.

OBJECTIVES: The present study characterized glial cell injury provoked in adult rat chiasm within 24 hours after a single, high-dose irradiation of 20 Gy. METHODS: All chiasmal glial cells in a section were counted, and the percentage of TUNEL-positive glial cells exhibiting apoptotic morphology was defined as the apoptotic rate. RESULTS: Numbers of apoptotic cells increased significantly (p<0.0001) from 3 to 8 hours after exposure, but returned to baseline levels by 24 hours. Little evidence of apoptosis was observed in non-irradiated chiasms. Similar patterns of increase in apoptotic rate were observed in the genu of the corpus callosum, but the extent was significantly lower (p=0.047) in the optic chiasm, with a maximal rate of 1.9%. Immunohistochemically, apoptotic cells were positive for CNP, a marker for oligodendrocytes. DISCUSSION: These data indicate that chiasmal irradiation induces limited, but significant apoptotic depletion of the oligodendroglial population, and may participate in the development of radiation-induced optic neuropathy.

Apoptosis and proliferation of oligodendrocyte progenitor cells in the irradiated rodent spinal cord.

PURPOSE: Oligodendrocytes undergo early apoptosis after irradiation. The aim of this study was to determine the relationship between oligodendroglial apoptosis and proliferation of oligodendrocyte progenitor cells (OPC) in the irradiated central nervous system. METHODS AND MATERIALS: Adult rats and p53 transgenic mice were given single doses of 2 Gy, 8 Gy, or 22 Gy to the cervical spinal cord. Apoptosis was assessed using TUNEL (Tdt-mediated dUTP terminal nick-end labeling) staining or by examining nuclear morphology. Oligodendrocyte progenitor cells were identified with an NG2 antibody or by in situ hybridization for platelet-derived growth factor receptor alpha. Proliferation of OPC was assessed by in vivo bromodeoxyuridine (BrdU) labeling and subsequent immunohistochemistry. Because radiation-induced apoptosis of oligodendroglial cells is p53 dependent, p53 transgenic mice were used to study the relationship between apoptosis and cell proliferation. RESULTS: Oligodendrocyte progenitor cells underwent apoptosis within 24 h of irradiation in the rat. That did not result in a change in OPC density at 24 h. Oligodendrocyte progenitor cell density was significantly reduced by 2-4 weeks, but showed recovery by 6 weeks after irradiation. An increase in BrdU-labeled cells was observed at 2 weeks after 8 Gy or 22 Gy, and proliferating cells in the rat spinal cord were immunoreactive for NG2. The mouse spinal cord showed a similar early cell proliferation after irradiation. No difference was observed in the proliferation response in the spinal cord of p53 -/- mice compared with wild type animals. CONCLUSIONS: Oligodendroglial cells undergo early apoptosis and OPC undergo early proliferation after ionizing radiation. However, apoptosis is not likely to be the trigger for early proliferation of OPC in the irradiated central nervous system.

Repopulation of oligodendrocyte progenitor cell depleted tissue in a model of chronic demyelination.

Some, but not all chronically demyelinated MS lesions are depleted of oligodendrocyte progenitor cells (OPCs) suggesting that OPCs are destroyed during the process of demyelination and some factor impedes OPC repopulation of the depleted tissue. The chronically demyelinated axons in MS lie in an astrocytic environment and it has been proposed that this might impede entry of OPCs into such regions. By depleting a short length of spinal cord of its OPCs using 40 Gy of X-irradiation in both normal rats and rats with progressive myelin loss accompanied by an astrocytosis (taiep rats), we investigated whether such changes affect the ability of OPCs to repopulate OPC-depleted tissue. In both taiep and normal rats, the rate of repopulation decreases with age, but no difference was detected in the rate at which OPCs repopulated normally myelinated and chronically demyelinated and astrocytosed tissue. This indicates that, if the astrocytic environment of the taiep CNS is comparable to that found in MS lesions, then the presence of chronically demyelinated axons and astrocytosis in chronic MS lesions does not represent a barrier to repopulation of the tissue by OPCs. However, similar to the situation in the normal adult rodent CNS, the rate of repopulation by endogenous OPCs in aged taiep rats is very slow, approximately 0.2 mm per week.

GLAST expression on bullfrog Müller cells is regulated by dark/light.

Using immunofluorescence double-labeling, Western blotting and confocal laser scanning microscopy, we investigated if and how the expression of glial high-affinity glutamate/aspartate transporter (GLAST) in bullfrog Müller cells may be regulated by dark/light. Compared with light-adapted retinas, the expression of GLAST in Müller cells was overall up-regulated in retinas dark-adapted for 30 min but declined in retinas dark-adapted for longer (>30 min) periods. The declined expression level of GLAST during prolonged dark adaptation was raised by immersion with 1 mM glutamate. These results suggest that glutamate uptake mediated by GLAST could be regulated dynamically and efficiently in accord with dark/light-induced changes in glutamate release of retinal neurons.

Astrocytes and oligodendrocytes express different STOP protein isoforms.

Many cell types contain subpopulations of microtubules that resist depolymerizing conditions, such as exposure to cold or to the drug nocodazole. This stabilization is due mainly to polymer association with STOP proteins. In mouse, neurons express two major variants of these proteins, N-STOP and E-STOP (120 kDa and 79 kDa, respectively), whereas fibroblasts express F-STOP (42 kDa) and two minor variants of 48 and 89 kDa. N- and E-STOP induce microtubule resistance to both cold and nocodazole exposure, whereas F-STOP confers microtubule stability only to the cold. Here, we investigated the expression of STOP proteins in oligodendrocytes and astrocytes in culture. We found that STOP proteins were expressed in precursor cells, in immature and mature oligodendrocytes, and in astrocytes. We found that oligodendrocytes express a major STOP variant of 89 kDa, which we called O-STOP, and two minor variants of 42 and 48 kDa. The STOP variants expressed by oligodendrocytes induce microtubule resistance to the cold and to nocodazole. For astrocytes, we found the expression of two STOP variants of 42 and 48 kDa and a new STOP isoform of 60 kDa, which we called A-STOP. The STOP variants expressed by astrocytes induce microtubule resistance to the cold but not to nocodazole, as fibroblast variants. In conclusion, astrocytes and oligodendrocytes express different isoforms of STOP protein, which show different microtubule-stabilizing capacities.

The infrared spectrum of human glioma cells is related to their in vitro and in vivo behavior.

The present research investigates whether infrared spectra can be related to the biological characteristics of glioma cell lines. We used nine human glioma cell lines for which a series of in vitro and in vivo biological features had already been established [Glia 36 (2001) 375] and were able to show that their characteristic infrared spectra reflect their in vitro migration (i.e., motility and invasiveness) properties and their in vivo aggressiveness. More particularly, the infrared data evidenced correlations at the level of the lipid/protein ratio. These relationships were found to be tissue-dependent when controlled on seven pancreatic carcinoma cell lines. We also showed that oligodendroglial and astrocytic tumor cells, whose identification remains difficult, can easily be identified by their infrared spectra in the lipid acyl chain region as well as in the nucleic acid region. We concluded that infrared spectroscopy could usefully complement information provided by more conventional diagnostic and prognostic (e.g., morphological and molecular) approaches.

Radiation-induced apoptosis of oligodendrocytes and its association with increased ceramide and down-regulated protein kinase B/Akt activity.

PURPOSE: Oligodendrocytes are cells responsible for myelination in the central nervous system and have been shown to undergo radiation-induced apoptosis. The roles of ceramide and protein kinase B/Akt (PKB/Akt) were assessed in radiation-induced apoptosis of oligodendrocytes in vitro. MATERIALS AND METHODS: Primary cultures of oligodendrocytes were established from neonatal rat brains and cell identity was assessed by immunohistochemistry. Apoptosis was assessed histologically according to its specific morphologic features using 4',6-diaminido-2-phenylindole, and by transferase-mediated deoxynucleotidyl transferase nick end-labelling staining. The ceramide level was measured using a diacyglycerol kinase assay, and PKB/Akt activity was determined using immunoblotting and a protein kinase assay. RESULTS: Ionizing radiation, C2-ceramide or wortmannin induced apoptosis in oligodendrocytes but not astrocytes. A rapid increase in ceramide was observed in oligodendrocytes after ionizing radiation. Monensin, an inhibitor of acid sphingomyelinase, reduced the apoptotic response in oligodendrocytes after ionizing radiation. Fumonisin B1, an inhibitor of ceramide synthase, showed no such effect in the cells. Radiation-induced apoptosis of oligodendrocytes was associated with a decrease in PKB activity, similar to that observed after treatment with C2-ceramide or wortmannin, but not after dihydro-C2-ceramide. Confocal microscopy revealed a loss of phosphorylated PKB immunostaining in the nucleus of apoptotic oligodendrocytes after ionizing radiation or C2-ceramide treatment. The level of phosphorylated FKHRL1, a transcription factor phosphorylated by PKB, decreased in irradiated oligodendrocytes. CONCLUSIONS: A ceramide-PKB-mediated signalling pathway might play a role in radiation-induced apoptosis of oligodendrocytes.