脑损伤

    From static to dynamic: live observation of the support system after ischemic stroke by two photon-excited fluorescence laser-scanning microscopy
  • Figure 2|Schematic of in vivo 2PLSM imaging for experimental ischemic stroke. 

    The first procedure for in vivo 2PLSM imaging of experimental ischemic stroke is the preparation of the cranial window for imaging in living animals. The thick and inhomogeneous structures of intact skulls cause severe optical aberrations and scattering noise, which greatly reduces image quality (Helmchen and Denk, 2005; Yoon et al., 2020). Thus, craniotomy or thinned-skull cranial window surgery using dental drills is needed (Xu et al., 2007; Marker et al., 2010; Yang et al., 2010). Different types of cranial windows are shown in Figure 2A–C. In general, craniotomy enables researchers to perform operations on the brain parenchyma, such as direct dye coverage and stereotactic injection of viruses or substances. Acute cranial windows can be combined with electrophysiology to measure neuronal electrical signals with electrodes simultaneously with 2PLSM imaging. Compared with acute or chronic craniotomy, the thinned-skull cranial window minimizes damage to brain tissue at the expense of imaging quality. Thinned-skull cranial window surgery is widely used, especially for imaging sensitive cells, such as microglia, or for structures that do not require high precision, such as imaging amyloid plaques (Xu et al., 2007; Yang et al., 2010). The second procedure for imaging of experimental ischemic stroke is visualization the components of interest in the brain, which is the selection of the fluorescent labeling strategies (Trachtenberg et al., 2002). A schematic of 2PLSM in vivo imaging of neurons and support systems is shown in Figure 2D. The fluorescent labeling strategies can be roughly divided into cell morphology and cell function labeling, namely, labeling the soma and processes to show cell morphology or labeling important intracellular functional ions, such as intracellular calcium, to show cell function. The commonly used fluorescent labeling methods are shown in Table 2 (Hartmann et al., 2015a; Hierro-Bujalance et al., 2018; Tong et al., 2021). The third procedure is the selection of specific imaging strategies using a 2PLSM system, including different excitation wavelengths different fluorescent labels as well as frame acquisition rates and timescales for different components. This aspect requires extensive experience and practical tests. The relevant parameters used are summarized in other articles (Benninger and Piston, 2013; Fumagalli et al., 2014; Ricard et al., 2018; Adhikari et al., 2021; Leben et al., 2022). The fourth procedure is inducing the experimental ischemic stroke. There are two commonly used experimental ischemic stroke models for in vivo 2PLSM imaging, middle cerebral artery occlusion (MCAO) and photothrombotic stroke (PT). A schematic diagram and images of in vivo 2PLSM imaging for MCAO and PT are shown in Figure 2E–H. MCAO is a widely used model of experimental ischemic stroke. To simulate stroke infarction and reperfusion, a single filament is introduced into the internal carotid artery from the external carotid artery and advanced to block the origin of the middle cerebral artery, blocking blood flow, and left in place for a period of time before being removed (Longa et al., 1989; Chiang et al., 2011). MCAO can form the ischemic penumbra, but the infarct area is large and the corresponding damage to the animals is also large (Sigler and Murphy, 2010; Krafft et al., 2012). In PT, the infarct is induced by the systematic application of a photosensitive dye (usually rose bengal) and illumination with light of a specific wavelength to block focal cortical blood flow (Kim et al., 2000; Labat-gest and Tomasi, 2013). This approach results in a rapid, stable, sharp-edged infarct suitable for studies of cortical plasticity; however, the infarct is permanent and lacks the ischemic penumbra (Nishimura et al., 2006; Zhang and Murphy, 2007; Krafft et al., 2012; Labat-gest and Tomasi, 2013; Li and Zhang, 2021). 

    Figure 3|Vessels observed by 2PLSM. 

    Vessel Dysfunction and Recovery after Ischemic Stroke Blood vessels, as pipelines for blood circulation, supply neurons and glial cells with essential nutrients, carry away metabolic waste, and form the barrier between the blood and cerebral parenchyma. In intact brains, blood vessels constitute a unique hierarchical 3D network (Kirst et al., 2020). Dysfunction and recovery in hemorheology, hemodynamics, and architecture of the vessel network significantly affect the pathological process of ischemic stroke and are associated with prognosis. In the acute phase of ischemic stroke, reperfusion therapy saves neurons in the ischemic penumbra and reduce mortality; this is regarded as the first-line therapy in ischemic stroke (Powers et al., 2019; Turc et al., 2019; Berge et al., 2021). However, timely and effective recanalization is not always accompanied by downstream tissue reperfusion, which has been described as the “no-reflow phenomenon” (Kloner, 2011; Bai and Lyden, 2015). Good collateral circulation is an independent predictor of improved outcomes and is a potential therapeutic target (Vernieri et al., 2001; Miteff et al., 2009; Shuaib et al., 2011; Vagal et al., 2018; Guglielmi et al., 2019; Broocks et al., 2020). In the recovery phase of ischemic stroke, angiogenesis in the peri-infarct region has been correlated with longer survival times in patients and coupled with neuronal remodeling (Kanazawa et al., 2019; Hatakeyama et al., 2020). Therefore, detailed intravital information on post-stroke hemodynamics, hemorheology, and vascular network architecture can help to gain a comprehensive understanding of the pathophysiological mechanisms of ischemic stroke and to develop corresponding therapies. By applying intravenous vascular tracers to label blood plasma, researchers can efficiently study the hemorheology, hemodynamics, and structure of hierarchical vessels (Figure 3A–C). Using transgenic fluorescent markers in animals, researchers can further image different cellular components of microvessel networks (Table 2). In this section, the network of vessels mainly refers to the cerebral microvessel system.

    Dynamic changes seen with embolisms in vivo are important because thromboses are the main initiator of ischemic stroke and are thought to be closely related to revascularization and prognosis (Powers et al., 2019; Turc et al., 2019; Berge et al., 2021). In 2010, using in vivo 2PLSM, Lam et al. (2010) first observed dynamic changes in microemboli in cerebral microvessels in living mouse brains. They used fluorescently labeled microemboli and found that embolus extravasation is an alternative recanalization mechanism when hemodynamic forces and the fibrinolytic system fail to clear the thrombus. The process of embolus extravasation occurs 2–7 days after thrombosis and is mediated by a novel mechanism of microvascular plasticity (Figure 3G; Lam et al., 2010). The rate of embolus extravasation is significantly reduced in aged mice or after inhibiting matrix metalloproteinase 2/9 activity (Lam et al., 2010). El Amki et al. (2020) induced a fibrin rich clot thrombin in the middle cerebral artery (MCA) followed by intravenous t-PA thrombolysis to mimic stroke and intravenous thrombolytic therapy, which differed from the classical MCAO model using filament to induce ischemia.They fluorescently labeled neutrophils, used in vivo 2PLSM to image the distal capillary flow after recanalization in mice and demonstrated that the no-reflow phenomenon after reperfusion therapy might be due to cortical microvascular occlusion caused by neutrophils (El Amki et al., 2020). These in vivo 2PLSM studies, using fibrin thrombi or cholesterol emboli, closely simulated the pathophysiological process of human ischemic stroke, which enabled researchers to dynamically observe changes in embolism in vivo with high spatiotemporal resolution. By monitoring blood flow in vivo after PT, Schrandt et al. (2015) tracked long-term vascular changes and found that a flow deficit remained even 35 days after occlusion, suggesting that more time was necessary for full perfusion recovery.

    Endothelial cells are indispensable components of the vasculature, and labeling endothelial cells rather than plasma can enable imaging of the whole vascular system without missing any capillaries (Williamson et al., 2020). Williamson et al. (2020) used transgenic mice expressing fluorescent proteins in endothelial cells to observe changes in the microvessel system after ischemic stroke (Figure 3D). Using in vivo 2PLSM and multi-exposure speckle imaging, they visualized the processes of structural vascular plasticity and the reconstruction of peri-infarct blood flow. They associated the rebuilding of blood vessels with neuronal network function recovery based on 2PLSM imaging of microvessels, multi-exposure speckle imaging of blood flow, and behavioral testing. They demonstrated that the extent of vascular structural plasticity predicted local blood flow reconstruction, which predicted the recovery of neuronal function.

    Figure 4|Calculation of BBB permeability.

    The BBB is a critical diffusion barrier between the brain parenchyma and cerebral capillaries that can prevent the influx of most toxic substances and provide essential nutrients (e.g., oxygen and glucose) to maintain brain homeostasis (Liebner et al., 2018; Vanlandewijck et al., 2018; Sweeney et al., 2019; Cheng et al., 2022). The BBB mainly comprises endothelial cells and tight junctions, astrocyte endfeet, and pericytes (Liebner et al., 2018; Sweeney et al., 2019; Profaci et al., 2020; Zou et al., 2021). To date, all of these components are considered to be indispensable to maintain the integrity and function of the BBB (Liebner et al., 2018; Caporarello et al., 2019; Heithoff et al., 2021). BBB dysfunction is commonly involved in the pathological processes that occur during the acute phase of ischemic stroke and is associated with ischemic stroke outcomes (Lasek-Bal et al., 2019; Li et al., 2019b; Bernardo-Castro et al., 2020). The integrity of the BBB is essential for maintaining homeostasis and supporting the function of neuronal and glial networks, which can be easily represented by permeability to fluorescent dyes during in vivo 2PLSM (see detailed information of calculation methods in Figure 4). In this section, we focus on the roles of endothelial cells and pericytes in the breakdown of the BBB and the roles of peripherally derived immune cells in ischemic stroke, while the role of astrocytes in maintaining the BBB is detailed in the section “Roles of communication between astrocytes and multiple cell types in the acute phase of ischemic stroke”. 

    Figure 5| Images from 2PLSM observation of astrocytes. 

    Ding et al. (2009) used in vivo 2PLSM imaging and found that, during the acute phase of PT, [Ca2+]i transients increased synchronously in astrocytes and propagated as waves in the astrocytic network. Using BAPTA to selectively inhibit [Ca2+]i transients in astrocytes could significantly reduce infarct volume. Additional studies have shown that IP3R2 receptor knockout mice and TRPV4 knockout mice, which exhibit reduced internal calcium release and external calcium inflow in astrocytes, respectively, also exhibited reduced glia-dependent glutamate release and performed better after experimental ischemic stroke (Ding et al., 2009; Dong et al., 2013; Rakers and Petzold, 2017; Rakers et al., 2017). Moreover, in aged mice, spontaneous Ca2+ activity in astrocytes and the infarct area were higher than those in adult mice after ischemic stroke, while spontaneous Ca2+ activity in neurons was unchanged (Figure 5A) (Fordsmann et al., 2019; Murmu et al., 2019). These studies showed that, after experimental ischemic stroke, [Ca2+]i increased in astrocytes, which may aggravate neuronal excitotoxicity through glia-dependent glutamate release (Ding et al., 2009; Dong et al., 2013; Rakers and Petzold, 2017; Rakers et al., 2017; Fordsmann et al., 2019; Murmu et al., 2019). Calcium waves in astrocytes and peri-infarct depolarizations are shown in Figure 5B. Shinotsuka et al. (2014) used acute cortical slices under OGD and showed differing results regarding the calcium wave in astrocytes. They found that the astrocytic gap junction network acted as a buffer for intercellular calcium fluctuations in neurons during the acute phase of ischemia. After blocking gap junctions between astrocytes in mouse cortical slices under OGD, the SD occurred earlier (Shinotsuka et al., 2014), showing a protective effect of astrocyte networks on neurons. However, in ex vivo studies, the severity of ischemia and hypoxia in brain tissue may differ from that in in vivo studies, affecting the roles of astrocytes after ischemia.

    The roles of astrocytes in the recovery phase of ischemic stroke are also complex. Based on postmortem techniques, Li et al. (2015) found that astrocyte IP3R2 knockout mice showed attenuated excessive astrogliosis and relieved brain injury, neuronal death, and behavioral deficits compared to controls 14 days after PT. This indicated that the calcium signal pathway in astrocytes may be harmful to neural recovery after ischemic stroke. However, other studies using dynamic in vivo 2PLSM showed that astrocytes played an essential role in angiogenesis, vascular network reorganization, and neural recovery. Heras-Romero et al. (2022) administered extracellular vesicles released by primary cortical astrocytes in the ventricles of mice and continuously observed the dynamic recovery process after ischemic stroke for 21 days. They found that the extracellular vesicles could mediate recovery of structure and function in neurons. Williamson et al. (2021) traced the morphology of astrocytes and the relationship between astrocytes and angiogenesis after PT for 28 days. They demonstrated that reactive astrocytes colocalized with and contacted newly formed vessels (Figure 5C). Chemogenetic ablation of peri-infarct reactive astrocytes dramatically impaired vascular remodeling and impeded the recovery of neurological function. G?bel et al. (2020) explored the specific mechanisms of astrocytes in vessel remodeling. They observed a prominent mitochondria-enriched compartment in astrocytic endfeet and mediation of vascular remodeling after stab-wound injury. However, this finding requires further study in ischemic stroke models.

    Figure 6|  Images of in vivo 2PLSM of microglia.  

    As a kind of non-excitable cell, microglia highly depend on changes in intracellular [Ca2+]i to perform cellular functions (Heo et al., 2015). Using in vivo two-photon calcium imaging, Eichhoff et al. (2011) found that most (80%) microglia showed no spontaneous Ca2+ transients at rest or under conditions of strong neuronal activity or intercellular astrocytic Ca2+ waves. However, microglia reliably responded with large, generalized Ca2+ transients to damage individual neurons (Eichhoff et al., 2011). This suggested that changes in microglial calcium signaling are primarily involved in pathological processes in response to neuronal injury, contrary to the results of in vitro experiments. Using in vivo 2PLSM, they found frequent Ca2+ transients in microglia after triggering cortical SD, which was induced by applying exogenous KCl solution (Tvrdik et al., 2019; Kearns et al., 2020, 2022; Liu et al., 2021). This linked neuronal damage, neuronal electrical signals, and microglial functional. A calcium wave in cortical microglia during ischemic stroke is shown in Figure 6B. This study also provided a reliable paradigm for studying the relationship between microglia and neuronal networks after ischemic stroke.
    Moreover, Cserép et al. (2020) used post hoc confocal laser scanning microscopy and electron microscopy to further illustrate the highly dynamic ultrastructure of microglia-neuron junctions in healthy brains, which provided valid structural evidence for the novel microglia-neuron junction concept (Figure 6A). They demonstrated that the microglia-neuron junctions consisted of closely apposed mitochondria, reticular membrane structures, intracellular tethers, and associated vesicle-like membrane structures within the neuronal cell body (Cserép et al., 2020). They used mitochondrial fluorescent labeling and in vivo 2PLSM to demonstrate that the foundation of microglia-neuron junctions relied on mitochondria-dependent neuronal exocytosis release signals (Cserép et al., 2020). This signaling process was highly correlated with ATP and ADP, which are important ligands for the regulation of microglial processes via the microglial purinoceptor P2Y12 (Cserép et al., 2020). They proposed that healthy neurons may constitutively release ATP and other signaling molecules at the microglia-neuron junction and that P2Y12 receptors on microglia receive the relevant signals, thus allowing microglia to monitor neuronal status (Cserép et al., 2020). Blocking microglial P2Y12 receptors after MCAO led to a strong increase in neuronal calcium load and to a significantly larger lesion volume than those observed in control mice, which may indicate that microglia play a neuroprotective post-stroke role through microglia-neuron junctions (Cserép et al., 2020).


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  • 发布日期: 2023-03-29  浏览: 79
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