Phenotype and Secretory Responses to Oxidative Stress in Microglia

Abstract

The neonatal brain is particularly susceptible to oxidative stress. Our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (H₂O₂) levels rise significantly particularly in the neonatal brain and are sustained for up to 7 days. This rapidly accumulated H₂O₂ is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. To date, there is limited literature on the effects of increased H₂O₂ levels on microglial cells, which have been extensively implicated in the ensuing inflammatory injury. Microglial cultures were derived from the P1 mouse brain and exposed to either bolus concentrations of H₂O₂ (15 or 50 μ<smlcap>M</smlcap>) or varying concentrations of continuous exposure for 4, 18 or 24 h. Continuous exposure of microglia to H₂O₂ was generated using the glucose oxidase-catalase system generating levels of H₂O₂ <10 μ<smlcap>M</smlcap>. Reactive oxygen species and nitric oxide expression were measured. Conditioned medium was collected and analyzed for secreted cytokine levels. Treated cell extracts were processed for glutathione (oxidized and reduced) content and fixed cells were labeled for M1 and M2a phenotype markers. Overall, it is evident that microglial exposure to continuous H₂O₂ has pleiotropic and biphasic effects. Continuous exposure to very low levels of H₂O₂ is more damaging to cell survival than higher bolus doses at 18 h, and can produce considerably high levels of pro- and anti-inflammatory cytokines by 18 h. Significantly high levels of various chemokines/chemotactic molecules such as G-CSF, MIP-1b and MIP-2 are also produced in response to continuous low-dose H₂O₂ by 18 h. Interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. H₂O₂ exposure promotes an M2a microglial phenotype in the absence of IL-4/IL-13 signaling, suggesting a wound-healing role for microglia and a delayed activation mechanism for H₂O₂ after such an insult. Together, these specific effects can be used to clarify the microglial cell responses following injury in the immature brain.

Temporal profile of M1 and M2 responses in the hippocampus following early 24 h of neurotrauma

“…An additional challenging factor is that some of the reactive components work in both directions. For example, exposure of hydrogen peroxide causes oxidative stress, but also helps in the expression of M2 phenotypes in microglia [60]. On the other hand, one treatment may influence different mechanisms, such as a toll-like receptor 2 agonist that induces a mixture of microglial polarization (both the M1 and M2 responses) [80].…”

The impact of high and low dose ionising radiation on the central nervous system

“…Activated microglia initiate an inflammatory response by releasing pro-inflammatory factors including cytokines and ROS [20], [31]. The pro-inflammatory state of activated microglia, or the M1 type classical activation state, can be cytotoxic to surrounding cells, and when unregulated can propagate tissue damage and cause secondary injury [32], [33]. Correspondingly, neuroinflammation is thought to be implicated in multifarious functions and pathological states in the CNS, including the modulation of neurogenesis and neuronal development [25], [34], [35], synaptic stripping and neuronal dysfunction [36], [37], and is now widely implicated in the pathogenesis and progression of many neurodegenerative disorders [38], [39], [40], [41], [42], [43], [44], [45].…”

Microglia toxicity in preterm brain injury

“…Continuous low H 2 O 2 also leads to significant production of pro-inflammatory cytokine IL-15, and chemokines ( e.g. granulocyte colony-stimulating factor, macrophage inflammatory protein-1 and macrophage inflammatory protein 2-alph) [89]. Activated microglia production of intracellular and extracellular ROS is dependent on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) [90–92].…”