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

Abstract

BackgroundIschemic 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 versus 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.MethodsMale 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 post-menopause. Brain tissue was collected to quantify infarct area and for immunohistochemistry and western blot methods. Ionized calcium-binding adapter molecule, TMEM119, and confocal microscopy were used to analyze microglia morphology and TMEM119 area in ipsilateral brain regions. Western blot was used to quantify protein quantity.ResultsPost-stroke injury is increased in male and female post-menopause mice versus pre-menopause female mice (p<0.05) with differences primarily occurring in caudal sections. After stroke, microglia underwent a region, but not sex group, dependent transformation into less ramified cells (p<0.0001). However, the number of phagocytic microglia were increased in distal ipsilateral regions of postmenopausal mice versus 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 TMEM119 immunofluorescence area decreased in proximity to the infarcted area, TMEM119 protein quantity was unchanged in ipsilateral hemisphere regions using western blot methods.ConclusionsOur 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.…”

“…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.…”

Plasticity of microglia

“…Various pathologies affect microglial expression of TMEM119 and hence impair its staining specificity (Young et al ., 2021). For example, in mice with ischemic stroke, TMEM119‐positive cells disappeared from nervous tissue far beyond the infarct border, while IBA1 staining was unaffected; furthermore, the pattern of expression of TMEM119 immunostaining was not consistent with western blot analysis used to validate immunohistochemistry.…”

“…For example, in mice with ischemic stroke, TMEM119‐positive cells disappeared from nervous tissue far beyond the infarct border, while IBA1 staining was unaffected; furthermore, the pattern of expression of TMEM119 immunostaining was not consistent with western blot analysis used to validate immunohistochemistry. While TMEM119 immunofluorescence decreased in the proximity of the infarct area, western blot of TMEM119 remained unchanged (Young et al ., 2021). In a traumatic brain injury model, surveilling microglia showed higher expression of TMEM119 as compared with reactive microglia, suggesting that TMEM119 immunostaining may be more suitable for identifying ramified microglia in both normal and injured brains (Abe et al ., 2018).…”

Quantitative evidence of suppressed TMEM119 microglial immunohistochemistry in fatal morphine intoxications

“…TMEM119 has served as a reliable immunohistochemical microglia marker in forensic assessments of traumatic causes of death, e.g., TBI [14], and was even positively stained by an adapted protocol for immunocytochemistry in postmortem CSF samples of trauma cases [15]. However, its function and involvement in the brain's injury response are yet to be defined [16].…”