The phenotypic distinction between CNS-infiltrating macrophages and brain-resident microglia is a major immunohistochemical concern. It is not only important in recognizing the origin of these cell populations, but it is also of fundamental importance in assessing the pathogenic and therapeutic significance of immune cells within the damaged brain. The lack of a single specific membranous and/or biochemical marker allowing definitive and discriminating identification of these cells is still a puzzling issue in neurobiology. Fortunately, alternative methods have been developed to overcome this hurdle, including the generation of bone marrow chimeric mice, which has proven to provide a powerful tool to distinguish microglia and infiltrated macrophages following ischemic stroke [
36,
37]. In this study, however, we made use of the CX
3CR1
+/GFPCCR2
+/RFP transgenic mouse model, which is based on the fact that chemokine receptor CX
3CR1 is predominantly expressed by CNS resident microglia and that CCR2 is upregulated in activated infiltrated macrophages and therefore allows distinction of eGFP-expressing microglia from RFP-expressing infiltrated macrophages [
5]. Through the use of this transgenic mouse model and immunofluorescence microscopy, we were able to demonstrate a region-dependent distribution of microglia and macrophages at day 14 after stroke. In the cortical ischemic lesion, CX
3CR1
+ microglia distinctly accumulated at the border of the lesion, while CCR2
+ macrophages were localized at the core of the lesion. At day 14, after ischemic stroke, CX
3CR1
+/GFP microglia were the major contributors of recruited cells to the ischemic lesion. These observations are in line with our earlier findings following MSC transplantation in mouse brain, which demonstrated a similar distinct distribution of microglia and macrophages, where infiltrated macrophages invaded the hypoxic/ischemic MSC transplant core, while active microglia remained in the surrounding border [
23,
25,
38]. In contrast to their orderly distribution in the cortex, both CX
3CR1
+ microglia and CCR2
+ macrophages were found to be randomly distributed in striatal lesions. In agreement with our own observations, others have also reported that resident microglia and blood-derived macrophages localize in the ischemic brain with different temporal and spatial patterns. Garcia-Bonilla et al. describe that at the first week after stroke, mostly diffused CCR2
+ macrophages were observed throughout the ischemic lesion, while during the second and third week after stroke, they saw that CX
3CR1
+ cells were localized in the peri-infarct area encircling the ischemic lesion core filled with CCR2
+ macrophages [
39]. Indeed, in a rather complex study design, these latter authors demonstrated that CCR2
+CX3CR1
− monocytes may, in a time-dependent fashion after infiltration, turn into CCR2
−CX3CR1
+ macrophages. Thus, we must caution that our assignment of green fluorescent, CX3CR1-positive microglia may to some extent also contain macrophages. This, however, does not affect our assignment of CCR2
+ red cells as a pure macrophage population. Another study showed that infiltrated blood-borne monocytes were exclusively located at the ischemically injured striatum at days 3, 7, and 14 after MCAO [
40]. Their results represent a random distribution of brain-resident microglia and infiltrated monocyte within the striatum after ischemic stroke, with microglia being the vast majority of cells invading the ischemic lesion. Moreover, in a mouse model of traumatic spinal cord injury, CX
3CR1
+/GFP and CCR2
+/RFP cells were randomly distributed around and inside the lesion, with CCR2
+/RFP cells constituting the greater number of accumulated cells in the lesion area [
41].
In our experimental setup, considering CX
3CR1
+/GFP cells as brain-resident microglia, it might be argued that perivascular macrophages, supraependymal macrophages, epiplexus cells of the choroid plexus, and meningeal macrophages express CX
3CR1 too [
44]. In addition, we cannot neglect the presence of patrolling macrophages Ly6c
− CX
3CR1
high CCR2
low in the ischemic brain with disrupted blood-brain barrier [
41,
45]. Interestingly, in an attempt to observe chemokine receptor expression changes in microglia and monocytes/macrophages in development and during inflammatory condition, Mizutani and colleagues have indicated that CCR2 is absent in adult naïve and inflamed CNS resident microglia [
5]. Importantly, they have demonstrated that CX
3CR1 expression by microglia is significantly higher compared to CX
3CR1
low CCR2
high or CX
3CR1
high CCR2
low monocytes/macrophages. Therefore, we hypothesize that CX3CR1
+/GFP cells are mostly of brain-resident microglial origin and double-positive cells for CX
3CR1
+/GFP and CCR2
+/RFP are blood-derived macrophage populations [
33,
41,
46]. It should be emphasized that the presence of double-positive cells at day 14 after ischemic stroke accounts for a very small proportion of infiltrated macrophages in our experiment. Further assessment of earlier or later time point of macrophage recruitment following CNS damage should also be evaluated. Nevertheless, the discovery of more microglia-specific markers would contribute to more efficient discrimination between CNS resident microglia and infiltrated macrophages in future studies.