Abstract
Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor.
Highlights
Microglia are brain-resident phagocytic cells, which originate from a population of myeloid progenitors from the yolk sac during embryonic development [16, 23, 35] and are maintained through self-renewal without influx of peripheral cells during adult life [1, 4]
Analyzing the results of these efforts, we here confirm that human microglia can be readily isolated from post-mortem central nervous system (CNS) tissue based on the membrane expression of CD11b, that microglia are distinguishable from macrophages, and that the yield of viable microglia is linked to the acidification of the CNS at time of autopsy
We have previously shown that macrophages isolated from choroid plexus (CP) are distinguishable from microglia by size, granularity, and CD45/CD11b expression [25, 26], these analyses were performed on separately isolated populations of cells
Summary
Microglia are brain-resident phagocytic cells, which originate from a population of myeloid progenitors from the yolk sac during embryonic development [16, 23, 35] and are maintained through self-renewal without influx of peripheral cells during adult life [1, 4]. Mizee et al Acta Neuropathologica Communications (2017) 5:16 prone state [22, 30], the main reasoning behind microglia being linked to pathology in neurodegenerative disorders such as Alzheimer’s disease (AD) [21], Parkinson’s disease (PD) [33], and multiple sclerosis (MS) [24] Their role as possible contributors to disease has been complemented by evidence for their involvement in the pathophysiology of developmental and psychiatric disorders, such as major depression disorder, bipolar disorder, schizophrenia, and autism [3, 7], either through modulation of neuroinflammation or neuronal plasticity. The isolation of microglia from the brains of various genetic mouse models has greatly facilitated our understanding of basic microglia characteristics in health and disease [9] These models are of limited value in relation to human CNS disorders. To investigate the role of microglia in human context it is crucial to study human primary microglia
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