Stage of Oligodendrocytes Determines Response to Microglia In Vitro
Stage of Oligodendrocytes Determines Response to Microglia In Vitro
Background: Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.
Methods: OPCs and microglia were isolated from rat cortex. OPCs were induced to differentiate into oligodendrocytes with thyroid hormone in defined medium. For selected experiments, microglia were added to OPC or oligodendrocyte cultures. Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFα ELISA. Cell survival was assessed with immunocytochemistry and cell counts. OPC proliferation and oligodendrocyte apoptosis were also assessed.
Results: OPCs and oligodendrocytes displayed phenotypes representative of immature and mature oligodendrocytes, respectively. Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes. Activated microglia also underwent cell death themselves.
Conclusion: Activated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival. This may be of importance because activated microglia are present in several disease states where both OPCs and mature oligodendrocytes are also reacting to injury. Activated microglia may simultaneously have deleterious and helpful effects on different cells after central nervous system injury.
Oligodendrocytes develop from a bipotential progenitor cell, often referred to as an oligodendrocyte progenitor cell (OPC), or oligodendrocyte - type 2 astrocyte cell (O2A), as it can differentiate into either an oligodendrocyte or astrocyte in vitro. Though more prevalent in the immature CNS, OPCs persist in the CNS of mature animals and humans, and have been shown to respond to CNS injury by proliferating and possibly taking the place of mature oligodendrocytes that are lost during injury or disease.
Both OPCs and oligodendrocytes die in a variety of CNS diseases. In periventricular leukomalacia (PVL), OPCs are lost as a consequence of hypoxia, ischemia, or intrauterine infection. This loss of OPCs, and the resulting failure to replace mature oligodendrocytes, is thought to be the pathologic cause of spastic cerebral palsy. Mature oligodendrocytes are lost in multiple sclerosis (MS), the most common disease of the adult CNS, affecting over 250,000 people living in the US. Oligodendrocytes and OPCs also die after CNS trauma, such as brain and spinal cord injury and it has been shown that oligodendrocyte apoptosis in experimental spinal cord injury peaks over a week after the initial insult. This delayed oligodendrocyte death may reduce the effectiveness of neural conduction in the spared axons that often exist after spinal cord injury.
CNS inflammation occurs in both disease and trauma, and is mediated in part by microglia, the resident immune cells of the CNS. Microglia originate from bone marrow and migrate into the CNS during early stages of development. Microglia display graded levels of activation in the CNS, from resting, highly ramified microglia, to phagocytic macrophages. Microglia react quickly in response to CNS injury or disease, migrating into an injury site and secreting a wide array of molecules that can be toxic to OPCs and oligodendrocytes, including tumor necrosis factor-α (TNFα), glutamate, and free radicals. Activated microglia contribute to OPC and oligodendrocyte death in models of PVL and MS. Furthermore, molecules that induce oligodendrocyte death can also lead to microglial activation, such as glutamate and proinflammatory cytokines.
in vitro, microglia are capable of inducing OPC death even without the two cell populations being in direct contact. However, in vivo microglia have been observed in close proximity to dying oligodendrocytes after spinal cord injury. This proximity after injury suggests a mechanism by which microglia may influence oligodendrocyte and OPC survival, as it has been shown in vitro that microglia in contact with oligodendrocytes can induce oligodendrocyte death via membrane-bound TNFα which is more potent than soluble TNFα. Additionally, any soluble factors secreted by microglia could have a higher effective concentration if secreted into a small space between cells.
There is also evidence that microglia may play a protective or helpful role in the injured CNS.In vitro, microglia can be recruited by soluble factors released by stressed oligodendrocytes, and support oligodendrocyte survival via insulin-like growth factor 2. Additionally, cytokines produced by microglia may aid in repair after injury, as mice lacking TNFα undergo delayed remyelination. Even the observations of Shuman and colleagues, that activated microglia are found in contact with apoptotic oligodendrocytes after spinal cord injury, raises the question of whether microglia destroy oligodendrocytes that would otherwise survive after injury, or are simply phagocytosing oligodendrocytes already destroyed by other toxins in the damaged CNS. Some data suggest that microglia play a dual role in CNS injury, exacerbating damage in some instances or at some times, and promoting repair or regeneration at others. Shuman and colleagues also reported that microglia undergo apoptosis after spinal cord injury. It has been demonstrated that certain types of toxin-induced microglial activation can result in microglial death both in vitro and in vivo and microglial death has also been described in concert with microglial activation in other in vivo injury paradigms.
The current studies were carried out to better to determine the effect of activated microglia on oligodendrocytes at different developmental stages and to assess microglial survival after activation. Though many studies have examined OPC and oligodendrocyte response to activated microglia, no study, to our knowledge, has directly compared the response of OPCs and oligodendrocytes to activated microglia under identical culture conditions. Examining OPC and oligodendrocyte survival under identical conditions is important, as these cell types are both present together in the injured CNS and may respond differently to the effects of microglial activation. In this study, we utilized lipopolysaccharide (LPS) to activate microglia. LPS activates Toll-like receptor 4 and causes microglia to release proinflammatory cytokines and become phagocytic. Studies in our laboratory showed that LPS induced TNFα release from microglia in a dose-dependent fashion (Figure 1). We found that LPS-activated, but not non-activated, microglia reduced OPC survival. However, both LPS-activated and non-activated microglia increased mature oligodendrocyte survival even as microglia themselves underwent activation-induced cell death. These findings suggest that oligodendrocytes at different developmental stages respond differently to activated microglia and that OPCs and mature oligodendrocytes may undergo different fates in the face of microglial activation in vivo.
(Enlarge Image)
TNFα production by microglia. In a preliminary experiment, microglia alone were treated with LPS at varying doses and TNFα production was assessed 24 hours later. LPS induced TNF? production in a dose dependent manner.
Background: Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.
Methods: OPCs and microglia were isolated from rat cortex. OPCs were induced to differentiate into oligodendrocytes with thyroid hormone in defined medium. For selected experiments, microglia were added to OPC or oligodendrocyte cultures. Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFα ELISA. Cell survival was assessed with immunocytochemistry and cell counts. OPC proliferation and oligodendrocyte apoptosis were also assessed.
Results: OPCs and oligodendrocytes displayed phenotypes representative of immature and mature oligodendrocytes, respectively. Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes. Activated microglia also underwent cell death themselves.
Conclusion: Activated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival. This may be of importance because activated microglia are present in several disease states where both OPCs and mature oligodendrocytes are also reacting to injury. Activated microglia may simultaneously have deleterious and helpful effects on different cells after central nervous system injury.
Oligodendrocytes develop from a bipotential progenitor cell, often referred to as an oligodendrocyte progenitor cell (OPC), or oligodendrocyte - type 2 astrocyte cell (O2A), as it can differentiate into either an oligodendrocyte or astrocyte in vitro. Though more prevalent in the immature CNS, OPCs persist in the CNS of mature animals and humans, and have been shown to respond to CNS injury by proliferating and possibly taking the place of mature oligodendrocytes that are lost during injury or disease.
Both OPCs and oligodendrocytes die in a variety of CNS diseases. In periventricular leukomalacia (PVL), OPCs are lost as a consequence of hypoxia, ischemia, or intrauterine infection. This loss of OPCs, and the resulting failure to replace mature oligodendrocytes, is thought to be the pathologic cause of spastic cerebral palsy. Mature oligodendrocytes are lost in multiple sclerosis (MS), the most common disease of the adult CNS, affecting over 250,000 people living in the US. Oligodendrocytes and OPCs also die after CNS trauma, such as brain and spinal cord injury and it has been shown that oligodendrocyte apoptosis in experimental spinal cord injury peaks over a week after the initial insult. This delayed oligodendrocyte death may reduce the effectiveness of neural conduction in the spared axons that often exist after spinal cord injury.
CNS inflammation occurs in both disease and trauma, and is mediated in part by microglia, the resident immune cells of the CNS. Microglia originate from bone marrow and migrate into the CNS during early stages of development. Microglia display graded levels of activation in the CNS, from resting, highly ramified microglia, to phagocytic macrophages. Microglia react quickly in response to CNS injury or disease, migrating into an injury site and secreting a wide array of molecules that can be toxic to OPCs and oligodendrocytes, including tumor necrosis factor-α (TNFα), glutamate, and free radicals. Activated microglia contribute to OPC and oligodendrocyte death in models of PVL and MS. Furthermore, molecules that induce oligodendrocyte death can also lead to microglial activation, such as glutamate and proinflammatory cytokines.
in vitro, microglia are capable of inducing OPC death even without the two cell populations being in direct contact. However, in vivo microglia have been observed in close proximity to dying oligodendrocytes after spinal cord injury. This proximity after injury suggests a mechanism by which microglia may influence oligodendrocyte and OPC survival, as it has been shown in vitro that microglia in contact with oligodendrocytes can induce oligodendrocyte death via membrane-bound TNFα which is more potent than soluble TNFα. Additionally, any soluble factors secreted by microglia could have a higher effective concentration if secreted into a small space between cells.
There is also evidence that microglia may play a protective or helpful role in the injured CNS.In vitro, microglia can be recruited by soluble factors released by stressed oligodendrocytes, and support oligodendrocyte survival via insulin-like growth factor 2. Additionally, cytokines produced by microglia may aid in repair after injury, as mice lacking TNFα undergo delayed remyelination. Even the observations of Shuman and colleagues, that activated microglia are found in contact with apoptotic oligodendrocytes after spinal cord injury, raises the question of whether microglia destroy oligodendrocytes that would otherwise survive after injury, or are simply phagocytosing oligodendrocytes already destroyed by other toxins in the damaged CNS. Some data suggest that microglia play a dual role in CNS injury, exacerbating damage in some instances or at some times, and promoting repair or regeneration at others. Shuman and colleagues also reported that microglia undergo apoptosis after spinal cord injury. It has been demonstrated that certain types of toxin-induced microglial activation can result in microglial death both in vitro and in vivo and microglial death has also been described in concert with microglial activation in other in vivo injury paradigms.
The current studies were carried out to better to determine the effect of activated microglia on oligodendrocytes at different developmental stages and to assess microglial survival after activation. Though many studies have examined OPC and oligodendrocyte response to activated microglia, no study, to our knowledge, has directly compared the response of OPCs and oligodendrocytes to activated microglia under identical culture conditions. Examining OPC and oligodendrocyte survival under identical conditions is important, as these cell types are both present together in the injured CNS and may respond differently to the effects of microglial activation. In this study, we utilized lipopolysaccharide (LPS) to activate microglia. LPS activates Toll-like receptor 4 and causes microglia to release proinflammatory cytokines and become phagocytic. Studies in our laboratory showed that LPS induced TNFα release from microglia in a dose-dependent fashion (Figure 1). We found that LPS-activated, but not non-activated, microglia reduced OPC survival. However, both LPS-activated and non-activated microglia increased mature oligodendrocyte survival even as microglia themselves underwent activation-induced cell death. These findings suggest that oligodendrocytes at different developmental stages respond differently to activated microglia and that OPCs and mature oligodendrocytes may undergo different fates in the face of microglial activation in vivo.
(Enlarge Image)
TNFα production by microglia. In a preliminary experiment, microglia alone were treated with LPS at varying doses and TNFα production was assessed 24 hours later. LPS induced TNF? production in a dose dependent manner.