Microglia are ramified, dynamic cells in the healthy brain capable of a variety of morphological responses during disease and injury [16, 21]. As such, much research is currently centered on elucidating the mediators of altered microglia activity, including both morphology and function, in the healthy brain as well as in models of central nervous system injury [10, 11, 13, 16, 21, 30, 31]. The total progressive morphological and functional changes occurring during ischemia and reperfusion that contribute to cerebral injury after ischemic stroke are poorly understood [2, 6, 8, 32, 33]. In the present study, we tested the hypotheses that ischemia and reperfusion would elicit differing microglia morphological responses and that a spatiotemporal relationship exists between microglia morphology and evolving brain injury after ischemic stroke and reperfusion. Quantitative analysis of microglia morphology and associated microglia proinflammatory function revealed two important results: (1) a spatiotemporal relationship exists between microglia morphology and evolving cerebral injury in the ipsilateral hemisphere after ischemic stroke and reperfusion and (2) altered microglia function, as measured by increased phagocytic marker CD11b expression, is observed in regions of hyper- and de-ramified microglia morphologies during the evolution of cerebral injury after ischemic stroke and reperfusion.
Distinguishing between microglia and systemic immune cell (macrophage and neutrophil) responses to injury and disease in situ is challenging as these cells share surface and intracellular markers [17, 18]. However, microglia’s arborized morphology and dynamic processes behavior allow for the discrimination between these cell populations. In this study, we show that iba-1-positive cells had distinctive arborized features after ischemia alone and after ischemic stroke and 8 h of reperfusion. From this observation we concluded that microglia, as opposed to infiltrating cells, are indeed first responders after ischemic stroke and reperfusion, a notion that is supported by recent research [20, 34]. Alternatively, the presence of infiltrating macrophages and neutrophils after ischemic stroke and 24 h of reperfusion cannot be ruled out given robust de-ramification and amoeboid-like morphology of a portion of iba-1 positive cells most proximal to the necrotic core.
Cerebral ischemia and ischemia coupled with reperfusion result in differing pathologic mechanisms, both of which contribute to the evolving brain infarct after stroke . In addition, the heterogeneity and related regional vulnerability of brain tissue to cerebral ischemia and ischemia with reperfusion also differentially contribute to brain injury after stroke [33, 35, 36]. Therefore, we studied the effects of cerebral ischemia and ensuing reperfusion on ipsilateral microglial morphology during stroke. Using an objective skeleton analysis method to assess morphology, we illustrate that microglia have pleomorphic responses to cerebral ischemia, which includes both hyper- and de-ramification, proximal and distal to the developing necrotic core, respectively. Microglia surveillance is limited to specific micro-domains and, unlike astrocytes, has not been shown to be connected through a broader cell-cell network [10, 11, 13, 37]. Therefore, it is not surprising that during initial and localized injury, microglial responses may be diverse, representative of varied inputs from specific micro-domains. Although not investigated here, altered neuronal signaling during ischemia, toxic ATP and glutamate concentrations in the developing necrotic core, and altered cell receptor expression may influence microglia ramification status [14, 16, 20]. While cell necrosis is localized to the necrotic core, distal regions are subject to spreading cortical depolarization, increased ATP release through astrocytic networks, altered neuronal signaling and variable tissue oxygenation, suggesting that regional exposure to different environmental cues may influence microglia morphological responses [19, 34, 35, 37].
While reperfusion after ischemic stroke is necessary to reduce ischemic injury, reperfusion also initiates a cascade of deleterious events that contributes to secondary brain injury known as reperfusion injury [8, 33]. In light of this, we quantified microglia morphology after 8 and 24 h of reperfusion, which would include the first phase of a biphasic opening of the blood–brain barrier as well as early and fully evolved brain lesions in a murine model [3, 26]. In contrast to the pleomorphic responses observed during ischemia, our findings suggest that reperfusion elicited only microglia de-ramification. Although striatal microglia were unchanged after ischemic stroke and 8 h of reperfusion, when compared to matching contralateral controls, process ramification was decreased when considering their initial hyper-ramified status. Microglia became incrementally de-ramified in the cortex, in a spatial relationship to the evolving injury, after 24 h of reperfusion and extended exposure to a deleterious milieu in the ipsilateral hemisphere. We expected a more widespread microglial response in the contralateral hemisphere in light of increased blood–brain barrier permeability [3, 38] and reports of microglia activation and phagocytic activity in the contralateral hemisphere following ischemic stroke [39, 40]. However, we showed that only contralateral striatal microglia but not cortical microglia were de-ramified when compared to comparable sham regions after ischemic stroke and 24 h of reperfusion. Although de-ramified in all ipsilateral regions when compared to their contralateral counterparts, microglia morphology remains diverse within the ipsilateral hemisphere during the first 24 h after reperfusion, an important observation as microglial function may be defined by varied degrees of damage after ischemic stroke [18, 41, 42]. Future studies addressing microglia morphology in response to their microenvironment are needed.
We conducted additional experiments in live cortical brain slices to further explore the spatial relationship between dynamic microglia morphology and cerebral injury after ischemic stroke and 24 h of reperfusion. Consistent with the fixed tissue data, microglia cells in an ex vivo slice preparation were de-ramified in relation to the necrotic core at the 24-h reperfusion time point. The incidence of active microglia was reduced in the ipsilateral hemisphere, and within the cohort of active cells, microglia process activity (protrusion/retraction) in the peri-infarct region was severely blunted when compared to the contralateral hemisphere. In agreement with previous studies [14, 34], we suggest that the microglia ramification status is an indicator of microglia process activity after ischemic stroke and reperfusion. Image acquisition of live microglia at 60× magnification was necessary to achieve sufficient resolution to detect microglia processes in the heterozygous CX3CR1-GFP mouse. We used the Sholl analysis methodology rather than the skeleton analysis approach for multiple reasons. Sholl analysis is ideally suited to the individual analysis of cell morphology rather than batch cell processing used in the skeleton analysis approach. In addition, the conversion of maximum intensity projection images to a binary and then skeletonized image resulted in inaccurate skeletonized structures, likely because of the bright soma of these cells and potential light scattering. Additional limitations were the slicing effects on microglia morphology  and bias of the experimenter in individual cell analysis. These limitations were addressed by acquiring live images within a 2-h window following brain slicing and acquiring images in deeper slice layers where live neurons and astrocytes are typically preserved and microglia are less affected by slicing procedure . While the tissue and cell morphology was distinct in region 2/peri-infarct, Sholl analysis of all other microglia cells was performed without knowledge of the brain region, decreasing bias. To our knowledge, this is the first study that applies the Sholl analysis to quantify microglia morphology. Nevertheless, we believe this to be an ideal analysis methodology for the live cells as the results yielded comparable data to that of the skeleton analysis.
In light of the novel identification of both hyper-ramified and de-ramified microglia subpopulations ipsilateral to cerebral injury after stroke, we endeavored to elucidate the role of microglia responses after ischemic stroke and reperfusion by studying associations between microglia form and function. Our investigation included iNOS and CD11b expression as markers of proinflammatory microglia function. Although therapeutic in the healthy brain, excessive NO production (mediated by iNOS) and phagocytosis (mediated by CD11b expression) contribute to secondary injury after ischemic stroke and reperfusion [2, 27, 43]. Multiple cell types, including microglia, contribute toward cytotoxic concentrations of NO via increased iNOS expression after ischemic stroke ; however, the presence of iNOS in ipsilateral brain tissue was not significantly increased until at least 72 h of reperfusion. The fact that we show distinct and diverse microglia morphologies that span the first 24-h period of ischemic stroke is suggestive that iNOS is not a suitable marker of early microglia functional response.
On the other hand, CD11b expression, a well-established marker of increased phagocytic and proinflammatory function [22, 29], is an excellent marker of early changes in microglia functional after cerebral ischemia and during early reperfusion. That microglia CD11b expression was significantly increased in regions of both hyper-ramified and de-ramified microglia further supports the notion that both hyper-ramified and de-ramified microglia should be considered when stratifying the microglial “activation” status during disease pathology. However, increased microglia CD11b expression was not exclusively associated with one type of microglia morphology. Thus, microglia phagocytosis may not be a general event but instead, influenced by differing environmental cues within microglia domains, is multi-purposed during spatiotemporal responses to ischemic stroke and reperfusion. For example, targeted phagocytosis directed by ramified microglia processes during cerebral ischemia may influence early synaptic remodeling and containment of developing cerebral injury , whereas de-ramified microglia, present after extensive and prolonged injury, contribute toward the clearing of diffuse cellular debris .