Can quantifying morphology and TMEM119 expression distinguish between microglia and infiltrating macrophages after ischemic stroke and reperfusion in male and female mice?

Abstract

Background Ischemic stroke is an acquired brain injury with gender-dependent outcomes. A persistent obstacle in understanding the sex-specific neuroinflammatory contributions to ischemic brain injury is distinguishing between resident microglia and infiltrating macrophages—both phagocytes—and determining cell population-specific contributions to injury evolution and recovery processes. Our purpose was to identify microglial and macrophage populations regulated by ischemic stroke using morphology analysis and the presence of microglia transmembrane protein 119 (TMEM119). Second, we examined sex and menopause differences in microglia/macrophage cell populations after an ischemic stroke. Methods Male and female, premenopausal and postmenopausal, mice underwent either 60 min of middle cerebral artery occlusion and 24 h of reperfusion or sham surgery. The accelerated ovarian failure model was used to model postmenopause. Brain tissue was collected to quantify the infarct area and for immunohistochemistry and western blot methods. Ionized calcium-binding adapter molecule, TMEM119, and confocal microscopy were used to analyze the microglia morphology and TMEM119 area in the ipsilateral brain regions. Western blot was used to quantify protein quantity. Results Post-stroke injury is increased in male and postmenopause female mice vs. premenopause female mice (p < 0.05) with differences primarily occurring in the caudal sections. After stroke, the microglia underwent a region, but not sex group, dependent transformation into less ramified cells (p < 0.0001). However, the number of phagocytic microglia was increased in distal ipsilateral regions of postmenopausal mice vs. the other sex groups (p < 0.05). The number of TMEM119-positive cells was decreased in proximity to the infarct (p < 0.0001) but without a sex group effect. Two key findings prevented distinguishing microglia from systemic macrophages. First, morphological data were not congruent with TMEM119 immunofluorescence data. Cells with severely decreased TMEM119 immunofluorescence were ramified, a distinguishing microglia characteristic. Second, whereas the TMEM119 immunofluorescence area decreased in proximity to the infarcted area, the TMEM119 protein quantity was unchanged in the ipsilateral hemisphere regions using western blot methods. Conclusions Our findings suggest that TMEM119 is not a stable microglia marker in male and female mice in the context of ischemic stroke. Until TMEM119 function in the brain is elucidated, its use to distinguish between cell populations following brain injury with cell infiltration is cautioned.

Transmembrane protein 119 is neither a specific nor a reliable marker for microglia

“…In line with our findings, a body of evidence has recently emerged indicating that (early) activated microglia significantly reduce TMEM119 expression (Cao et al, 2021;Deczkowska et al, 2018;Marzan et al, 2021;Masuda et al, 2020;van Wageningen et al, 2019;Young et al, 2021). Masuda and colleagues, for example, performed extensive single-cell RNA-sequencing and mass cytometry to describe the microglial core genes.…”

“…In the physiological CNS, a distinction between microglia and monocytes/macrophages is possible based on the different morphologies of both cell types. Microglia exhibit a highly ramified cell shape under physiological conditions, while monocytes and macrophages exhibit a constant amoeboid form (Morrison & Filosa, 2013;Young et al, 2021).…”

“…Although microglia are always in an active, monitoring state under physiological conditions, pathological changes within the CNS induce a complex cell-differentiation process, in which microglia cells retract their ramified processes and acquire an amoeboid form (Davalos et al, 2005;Guerrero & Sicotte, 2020;Nimmerjahn, Kirchhoff, & Helmchen, 2005;Young et al, 2021). A morphological distinction between the brain resident microglia and peripheral macrophages becomes now highly challenging.…”

Protein Expression of the Microglial Marker Tmem119 Decreases in Association With Morphological Changes and Location in a Mouse Model of Traumatic Brain Injury

“…However, the validity of Tmem119 as a microglia marker after TBI is yet to be confirmed. Recently, Young et al (2021) evaluated the expression of Tmem119 to distinguish between microglia and infiltrating macrophages in a mouse model of ischemic stroke. They reported that Tmem119 presence was decreased in ramified microglia in a brain region proximal to the site of injury, thus, concluding that Tmem119 is not a stable microglia marker in the context of ischemic stroke.…”

Flow cytometry analysis of immune and glial cells in a trigeminal neuralgia rat model

“…However, our experiments also had some limitations. Although TMEM119 has been identified as a surface marker of microglia that can be used to reliably distinguish microglia from infiltrating macrophages 24 , recent studies also reported that the expression of TMEM119 is not restricted to microglia in inflammation 25 , 26 . In our study, there were few Iba1/TMEM119-positive cells in the TREZ, so we still could not well distinguish between microglia and macrophages through the two cell markers by immunohistochemistry.…”