The ischemic environment drives microglia and macrophage function

Abstract

Cells of myeloid origin, such as microglia and macrophages, act at the crossroads of several inflammatory mechanisms during pathophysiology. Besides pro-inflammatory activity (M1 polarization), myeloid cells acquire protective functions (M2) and participate in the neuroprotective innate mechanisms after brain injury. Experimental research is making considerable efforts to understand the rules that regulate the balance between toxic and protective brain innate immunity. Environmental changes affect microglia/macrophage functions. Hypoxia can affect myeloid cell distribution, activity, and phenotype. With their intrinsic differences, microglia and macrophages respond differently to hypoxia, the former depending on ATP to activate and the latter switching to anaerobic metabolism and adapting to hypoxia. Myeloid cell functions include homeostasis control, damage-sensing activity, chemotaxis, and phagocytosis, all distinctive features of these cells. Specific markers and morphologies enable to recognize each functional state. To ensure homeostasis and activate when needed, microglia/macrophage physiology is finely tuned. Microglia are controlled by several neuron-derived components, including contact-dependent inhibitory signals and soluble molecules. Changes in this control can cause chronic activation or priming with specific functional consequences. Strategies, such as stem cell treatment, may enhance microglia protective polarization. This review presents data from the literature that has greatly advanced our understanding of myeloid cell action in brain injury. We discuss the selective responses of microglia and macrophages to hypoxia after stroke and review relevant markers with the aim of defining the different subpopulations of myeloid cells that are recruited to the injured site. We also cover the functional consequences of chronically active microglia and review pivotal works on microglia regulation that offer new therapeutic possibilities for acute brain injury.

Keywords: acute brain injury; cell morphology; macrophages; microglia; neuroinflammation; phenotypical polarization.

Figure 1

Microglia and macrophage distribution over the lesion area 24 h after injury in three different models of acute brain injury. (A) After transient middle cerebral artery occlusion (tMCAo), the lesion core is populated by infiltrated macrophages (M) and bushy/ameboid microglia (μ). The penumbra contains hypertrophic μ. (B) After permanent middle cerebral artery occlusion (pMCAo) no μ cells are present in the lesion core where there is high M infiltration. Bushy μ cells surround the lesion core. (C) The distribution is similar after traumatic brain injury (TBI). M are recruited to the lesioned tissue close to its lesion edge, while bushy and hypertrophic μ cells populate more distant areas.

Figure 2

CD11b or CD45 label myeloid cells in the mouse brain 24 h after focal transient ischemia. Left panel: CD11b is commonly used to label myeloid cells in the mouse brain and provides detailed information on morphology because it is expressed uniformly on the cell membrane. Twenty-four hours after focal transient ischemia, different microglia cell types can be found in the hemisphere ipsi-lateral to the lesion. Bushy microglia (μ) populate the cortex (penumbra) and rod microglia (rμ) with elongated morphology are present in the white matter (corpus callosum) and hypertrophic reactive microglia (reactive μ) populate the striatum, the area that corresponds to the lesion core. Right panel: CD45 labeling helps distinguish resident microglia from recruited leukocytes. Microglia have ramified morphology and pale staining (CD45^low), whereas infiltrated leukocytes (white blood cells, WBC) can be identified by their round-shaped morphology and strong, well-contrasted staining (CD45^high). The elongated shape of CD45^high cells may depend on crawling along vessel walls or their perivascular location. Bars = 100 μm, microphotograms modified from Ref. (40). Neuro-anatomical structures are indicated by CX (cortex), STR (striatum), CC (corpus callosum), and LV (lateral ventricle).

Figure 3

Evolution of myeloid cell morphology and phenotype in the brain during the acute phases of brain injury. Under physiological conditions, monocytes, microglia and rod microglia populate specific compartments, respectively blood, gray matter and white matter. After an insult to the brain, monocytes, microglia, and rod microglia are activated. Monocytes migrate to lesioned areas, whereas gray matter microglia are less likely to displace. Microglia change morphology in a time-dependent fashion, sprouting new ramifications soon after injury, and then withdrawing branches to develop an ameboid phenotype. Both infiltrated monocytes and microglia change their phenotypical profile, acquire specific functional commitments, and proliferate with time. In the very early phases after injury, microglia have a M1 phenotype, then, with the recruitment of macrophages, both myeloid populations upregulate M2 markers. The peak of M2 marker expression soon vanishes and is followed by upregulation of M1 markers that lasts longer. In vivo, mixed polarization phenotypes are observed and the M1-M2 definition may only serve to set the extremes of a continuum of polarization states. Macrophages from blood-borne monocytes seem to be more able to express M2 markers than microglia. In the white matter, rod microglia activate after injury by enhancing their elongated morphology, expressing reactive markers (CD68, MHCII), and migrating to other areas, such as the hippocampus. Rod microglia also cluster into trains of cells that align to neuronal axons. Neither the exact phenotype profile nor the functions of activated rod microglia are fully understood, though a role of these cells in synaptic stripping due to their contacts with axons has been hypothesized.

Figure 4

Shape changes of different myeloid cell types over time. Cell size (left panel) can be measured by assessing cell area or cell width (caliper). While in monocytes (M), the size is constant, microglia (μ) grow larger (hypertrophic reactive morphology), and rod microglia (rμ) first grow and then shrink (elongated not ramified morphology). Elongation (center panel) can be evaluated from the ratio between the cell’s major and minor axes. A certain degree of elongation is selectively associated with M switch to M2 polarization. Elongation of μ might occur during early damage-sensing as μ direct ramifications toward the site of injury. Then, along with ameboid transformation, μ recover their symmetric shape. In contrast, rμ progressively elongates so as to align with axons. The rate of branching (right panel) can be measured by counting the number of ramifications or by Sholl analysis. M does not have ramifications, while μ or rμ withdraw branchings over time to acquire, respectively, the ameboid or the elongated morphology: μ show a transient increase of ramifications early after injury that may be related to damage-sensing activity. Shape changes have been plotted according to the data and observations described in Ref. (40, 118, 160, 161, 164, 165).