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Updated: Aug 9, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
A serum factor enhances production of nitric oxide and tumor necrosis factor-alpha from cultured microglia
1Department of Physiology, Ehime University School of Medicine, Shigenobu, 791-0295, Japan.
Abstract:
The pathological activation of microglia has been implicated in ischemic neuronal damage and some neurodegenerative diseases; however, the mechanism of microglial activation is not well understood. Previously, we showed that a serum factor, albumin, increased O(2)(-) production by cultured microglia (Si et al., 1997, Glia 21: 413-418). In the present study, we found that serum also enhanced lipopolysaccharide (LPS)-induced production of nitric oxide and tumor necrosis factor-alpha, which are other important neurotoxins released by activated microglia. In the presence of 0.1% normal rat serum, the half-effective concentration for LPS decreased from 300 to 1 ng/ml. The factor seemed to be a relatively high-molecular-weight protein because the factor was retained after a molecular sieve (50 kDa) membrane separation. The factor was labile to trypsinization and heat treatment at 72 degrees C for 5 min but was stable at 56 degrees C for 60 min. Several purified serum proteins including albumin could not mimic the enhancing effect of serum. Acute-phase serum showed a potent enhancing effect at a 10 times lower concentration than the normal serum. By gel filtration chromatography, the enhancing effect observed was a single peak at about 60 kDa. These results suggest that some serum protein infiltrates into brain parenchyma after blood-brain barrier disruption and such protein may result in neuronal damage by activating microglia to release neurotoxins in some central nervous system diseases.
Insights
Serum factors enhance microglial activation, increasing neurotoxin release implicated in brain damage. This suggests proteins crossing a disrupted blood-brain barrier may drive neuroinflammation in central nervous system diseases.
Area of Science:
- Neuroscience
- Immunology
- Pathology
Background:
- Pathological microglial activation contributes to neuronal damage in ischemic and neurodegenerative conditions.
- The precise mechanisms driving microglial activation remain incompletely understood.
- Previous research indicated serum albumin increases microglial superoxide production.
Purpose of the Study:
- To investigate the role of serum factors in modulating microglial activation.
- To identify serum-derived mediators that enhance microglial release of neurotoxins.
- To explore the implications of these findings for central nervous system diseases.
Main Methods:
- Assessing lipopolysaccharide (LPS)-induced nitric oxide and tumor necrosis factor-alpha production by microglia in the presence of serum.
- Utilizing molecular sieve membrane separation (50 kDa) to characterize the molecular weight of the active factor.
- Employing trypsinization and heat treatment (72°C for 5 min, 56°C for 60 min) to assess factor stability.
- Testing purified serum proteins, including albumin, for their enhancing effects.
- Analyzing serum from acute-phase conditions.
- Performing gel filtration chromatography to isolate the enhancing factor.
Main Results:
- Normal rat serum significantly potentiated LPS-induced microglial neurotoxin production, lowering the effective LPS concentration.
- The active serum factor exhibited characteristics of a high-molecular-weight protein, retained by a 50 kDa membrane.
- The factor was sensitive to trypsin and heat (72°C) but stable at 56°C.
- Purified albumin did not replicate the serum's enhancing effect.
- Acute-phase serum demonstrated a markedly stronger enhancing effect than normal serum.
- Gel filtration identified a single peak of enhancing activity around 60 kDa.
Conclusions:
- Serum proteins infiltrating the brain parenchyma following blood-brain barrier disruption can activate microglia.
- This microglial activation leads to the release of neurotoxins, potentially causing neuronal damage.
- These findings suggest a mechanism linking blood-brain barrier dysfunction to neuroinflammation and damage in CNS diseases.
