Analysis Of Microglia Research Articles

Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions

Ex vivo analysis is an accurate and convenient way to study in vivo microglia phenotype and function. However, current microglia isolation protocols for ex vivo analysis show many differences in isolation steps (perfusion, removal of meninges and blood vessels, mechanical dissociation, enzymatic dissociation, density separation, immunomagnetic separation, and fluorescence-activated cell sorting), often without addressing their effects on microglia purity, number, phenotype, and function. Therefore, the aim of this study was to provide an optimized isolation protocol with emphasis on microglia purity and number to enable ex vivo analysis of adult mouse microglia. The application of this protocol for ex vivo phenotype and functional analysis is corroborated by results from flow cytometry, gene expression analysis, chemotaxis, and phagocytosis assays. In addition, this study shows the possibility to analyze microglia isolated from various central nervous system regions such as optic nerve, striatum, hippocampus, spinal cord, cerebellum, and cerebral cortex. Furthermore, this is the first study presenting DRAQ5 as a superior alternative to propidium iodide for the discrimination between living and dead cells. DRAQ5 staining facilitated the identification of microglia upon flow cytometry without the need of additional fluorescent markers. Along with a favorable emission spectrum, DRAQ5 proved a valuable tool for flow cytometry of microglia. The presented optimized microglia isolation protocol for ex vivo analysis offers the opportunity to obtain more insight into both general and region-specific microglia behavior.

Chemotherapy Delays Progression of Motor Neuron Disease in the SOD1 G93A Transgenic Mouse

Background: A significant proliferation of glial cells occurs in the spinal cord and brainstem of SOD1 G93A transgenic mice with familial amyotrophic lateral sclerosis (ALS). Since activated glia may contribute to motor neuron degeneration, we tested whether inhibition of gliosis using low-dose chemotherapy is beneficial in this mouse model. Methods: Mice were administered fortnightly intraperitoneal injections of 0.1 mg/kg vincristine (VIN) or saline commencing at postnatal day 68 before disease onset. Mice were sacrificed at end-stage disease, and spinal cords were examined for histology. Results: Survival of VIN-treated mice was significantly increased at 132.0 ± 4.1 days compared to control animals at 117.8 ± 2.1 days (p < 0.05). Furthermore, analysis of microglia and astrocyte populations suggests a reduction in the former following VIN therapy. Conclusion: This study suggests that chemotherapy may offer an alternative therapy or co-therapy for ALS.

Prion protein-overexpressing cells show altered response to a neurotoxic prion protein peptide.

A peptide fragment of the prion protein, PrP106-126 is toxic to neuronal cells in culture. This toxicity is dependent on neuronal expression of the prion protein (PrPc) and also the presence of microglia. The role of expression of the PrPc in neurotoxicity of this peptide was investigated using mice that overexpress the prion protein. Cells derived from two different strains of PrPc-overexpressing mice were used (Tg20 and Tg35). PrP106-126 was more toxic to Tg35 cerebellar cells than wild-type or Tg20 cells. This increased toxicity required the presence of microglia. Analysis of microglia derived from wild-type and PrPc-overexpressing cells showed that Tg35 microglia were more easily activated than wild-type microglia, were more easily stimulated to proliferate by astrocytes, and had a higher level of PrPc expression. This may explain the increased PrP106-126 toxicity to Tg35 PrPc-overexpressing cerebellar cells. These results suggest that the toxicity of PrP106-126 may depend on the level of expression of PrPc by microglia as well as by neurones.