Rapid isolation and culture of primary microglia from adult mouse spinal cord

Original article Effects of hypothermia on ex vivo microglial production of pro- and anti-inflammatory cytokines and nitric oxide in hypoxic-ischemic brain-injured mice

“…Importantly, as mentioned above, in terms of the temporal changes of neuroinflammatory responses, our present ex vivo findings, as well as our previous in vitro findings [33][34][35][36], appear to be similar to the in vivo findings for brain injury [9,11], as well as to those regarding IL-10 in vivo, which indicate that the levels in the brain show a peak during the first days (24-48 h) after brain injury [6,14,25]. Temporal similarity between the ex vivo and in vivo expressions of a signalling molecule in microglia has also been reported [12,51]. Thus, our ex vivo experiments with TLR stimulation may also allow us to study microglial function in pathophysiological states and therefore be useful in examining the effects of hypothermia.…”

“…Moreover, an investigation of the responses of microglia derived from brain-injured animals to TLR stimulation, along with their responses to hypothermia, is of particular significance because both microglial TLR2 and TLR4 play an important role in triggering immediate responses and/or enhancing reactions to tissue injury and inflammation [5,22,28]. In addition, an ex vivo culture of microglia isolated from CNS preserves the in vivo phenotype of microglia [18,51]. Importantly, as mentioned above, in terms of the temporal changes of neuroinflammatory responses, our present ex vivo findings, as well as our previous in vitro findings [33][34][35][36], appear to be similar to the in vivo findings for brain injury [9,11], as well as to those regarding IL-10 in vivo, which indicate that the levels in the brain show a peak during the first days (24-48 h) after brain injury [6,14,25].…”

“…An ex vivo setting was chosen as it more closely reflects in vivo conditions compared with cell-culture systems in vitro [18,44], and the cultures of ex vivo microglia obtained directly from the central nervous system (CNS) show functional and temporal similarities in the responsiveness of these cells in vivo to a stimulus [12,51]. Moreover, while TLRs are major sensors of pathogen-associated molecular patterns that mediate innate immunity and are involved in the adaptive immune response [2], they can also be stimulated by the non-physiological appearance or unusual concentrations of certain endogenous molecules [24], which may be produced and released by damaged cells in CNS.…”

A new method to isolate microglia from adult mice and culture them for an extended period of time

“…This isolation methodology has been improved and optimised with the addition of fluorescence activated or immunomagnetic cell sorting steps (Ford et al, 1995;Guillemin et al, 1997;De Groot et al, 2000;Frank et al, 2006;Cardona et al, 2006;De Haas et al, 2007). Recently, Yip et al (2009) described a simpler method based on the high adherence of microglia which allows the isolation of ex vivo adult primary microglia without gradient density centrifugation step. However, these different methods are limited to the direct use of the collected cells, for example for RNA or protein extraction (Cardona et al, 2006) or for steady state observation by flow cytometry (Ford et al, 1995).…”

Advantages and Pitfalls in Experimental Models Of ALS

“…Now, concerning astroglia culture, "primary culture", "subculture" and "shaken culture (once or twice)" can be performed (Du et al, 2010), each one with advantages and pitfalls relative to purity of astrocytes, cell viability, expression of glial fibrillary acidic protein (GFAP) and bystin, a protein potentially involved in embryo implantation, which is markedly up-regulated in reactive astrocytes (Fang et al, 2008). Similarly, protocols to isolate microglial cells are easy and reproducible (Ni & Aschner, 2010;Yip et al, 2009), and they have been recently improved by the column-free magnetic separation technology: the cells can be labeled and isolated on the strength of their expression of CD11b, a specific microglial marker, thus allowing to isolate an high number of cells and significantly reducing the animals needed (Gordon et al, 2011). Cocultures of healthy hMNs with human astrocytes carrying either the WT or mutated SOD1 cDNA have demonstrated the role of astrocytes in ALS disease, since MN number decreased about 50% in the presence of mutant SOD1-expressing astrocytes (Marchetto et al, 2008).…”