Ischemic stroke and cerebral infarction result from insufficient blood and oxygen supply to the brain. Blockage of the blood supply to the brain causes a cascade of pathological changes associated with energy failure, excessive glutamate release, elevated intracellular Ca²⁺ levels, generation of free radicals, blood-brain barrier (BBB) disruption, inflammation, and eventually massive cell death via necrosis and apoptosis [1]. The cells in the central portion of the ischemic tissue, also known as the infarct core, have been known to be afflicted by irreversible damage, and the cells around the infarct core, called the penumbra, also undergo delayed cell death within several hours or days [2,3].

Over the past several decades, the development of imaging technology, including magnetic resonance imaging (MRI), has allowed the visualization of temporal and spatial changes in brain lesions [4,5]. Many studies related to stroke lesions have revealed pathophysiological changes in the brain, including cell death-related pathways; however, most studies have been limited to specific times and areas.

In this study, we aimed to analyze the spatiotemporal expression profiles of cell death-related molecules in a commonly utilized rat stroke model of transient occlusion and reperfusion of middle cerebral artery (MCAO/R). We obtained MR and histological images from the same animals to minimize potential variances. In addition to microscopic images of the most representative regions, we provide the entire brain images with high resolution in the Supplementary Figures, in which the region of interest can be magnified to microscopic levels. This study showed that the pathophysiological changes in the whole brain including the penumbra and core at a specific time after an ischemic injury may provide useful information for determining the optimal administration route and therapeutic time window for diverse therapies.

Focal cerebral ischemia was induced by 2-h occlusion of the MCA and reperfusion. MRI tests immediately followed by TTC staining were carried out to evaluate the infarction. Animals with 0 h reperfusion following a 2 h occlusion showed no obvious infarct in both MRI and TTC tests. The infarct became evident in the striatum at 2 h after the reperfusion (4 h after the onset of ischemia) and gradually increased to the entire area of the striatum and dorsolateral cortex at 6 h. This condition is consistent with previous reports, which demonstrated that the striatum is more vulnerable to death in this stroke model because it is supplied by the lenticulostriate artery, a deep branch of MCA, whereas the cortex is supplied by MCA branches and collateral circulation on the border [7]. The severity of ischemic injury was highest between day 1 and 3 and apparently reduced from day 7 onwards (Fig. 1A). This decrease in infarct size can be attributed to infiltration of CD68-positive cells in the ischemic core, since TTC staining reveals the mitochondrial respiratory enzymes in all cell types (see Fig. 3).

After the TTC staining, the brain slices were fixed and embedded in the paraffin. Several 5 µm-thick paraffin sections were obtained and subject to histological analyses. In the low magnification of full-sized brain images in the H&E staining, the infarct was first detected at 2 h in the striatum and then in the overlying dorsolateral cortex at 6 h after reperfusion. However, microscopic analysis of the same brain slice revealed that degeneration actually occurred at 0 h (just after reperfusion) in the striatum and 2 h after the reperfusion in the cortex, as evidenced by darkly stained pyknotic nuclei, cell body shrinkage, and perineuronal vacuolization (arrows in Fig. 1B). At day 3, as the darkly stained nuclei increased in number, the neuropil in the background was more vacuolized and decomposed (Fig. 1B). On day 7, the area of ischemic core became hypercellular and packed with infiltrating immune cells, while the surrounding neuropil adopted a sponge-like appearance with a cavity (Fig. 1B). Tissue clearance of the ischemic core further progressed and the growing cavity areas remained at 1 month (Fig. 1B).

Immunostaining with the anti-NeuN antibody showed perinuclear immunoreactivity of the sham-operated animals (Fig. 1C). NeuN staining revealed that neuronal degeneration began at 0 h in the striatum and at 2 h in the cortex. NeuN immunoreactivity was changed in the ischemic core similar to that observed in the H&E staining until day 3 (Fig. 1C). Between day 7 and the end of 1 month, NeuN immunoreactivity completely disappeared in the ischemic tissues. The results are consistent with the previous finding that striatal infarction is mostly necrotic and occurs rapidly, while cortical infarction is more delayed and contains a greater degree of apoptotic cell death than observed in the striatal infarction [7,8].

GFAP immunostaining showed the typical staining of stellate, branched astrocytes in the sham group (Fig. 2). At 2 h after reperfusion, GFAP immunoreactivity began to decrease in the core region and eventually disappeared on day 7. Such rapid astrocytic death has been previously reported [9]. In the penumbra of the cortex, the GFAP immunoreactivity increased from 6 h and exhibited hypertrophied morphology between day 1 and 3. At day 7, astrocytes in the penumbra of the cortex were densely packed and formed a stream with elongated (straight) processes pointing toward the ischemic core, i.e., a feature of astroglial scar formation. At 1 month, the astrocytes exhibited overlapping processes in the compact scar border, suggesting the maturation of astroglial scar tissue (Fig. 2).

Activated microglia have been well established to transform into phagocytic cells that release a variety of cytotoxic substances following brain injury, which subsequently induce infiltration of circulating lymphocytes and macrophages [10]. In the sham group, Iba1-immunostaining showed resting microglia with highly branched, thin, and elongated processes (Fig. 3A). Within 1 day after ischemic injury, microglia underwent morphological changes to reach an activated amoeboid state in the core and penumbra rather than just an increase in number (Fig. 3B). In the subacute phase (days 3~7), most microglia in the lesion sites were strongly activated with round, amoeboid shapes, which are indistinguishable from those of infiltrating macrophages. The mixed microglia/macrophage population indicates the breakdown of BBB and massive infiltration of circulating immune cells. At 1 month, microglia were predominantly found in the penumbra as Iba1-positive cells (Fig. 3A). Quantitative analysis indicated that the Iba1 expression in the penumbra prominently increased from day 3, peaked at day 7, and then gradually declined at 1 month (Fig. 3B).

CD68 is a common marker for reactive microglia and macrophages [11]. Immunostaining with anti-CD68 antibody further supported our finding of microglial activation in the core and penumbra (compared in Fig. 3A and C). Unlike Iba1-staining, CD68 immunoreactivity was barely detectable in the sham-operated animals but became evident at 2~6 h after reperfusion in all affected areas, where Iba1-positive microglia displayed amoeboid morphology (Fig. 3C). In the subacute phase (days 3~7), CD68-positive cells appeared as a mixed microglia/macrophage population in the core and penumbra. Quantitative analysis indicated that the number of CD68-positive cells steadily increased up to day 7 in the core and penumbra (Fig. 3D). At 1 month when most ischemic tissues dissolved, the number of CD68-positive cells decreased to the level similar to that at 6 h (Fig. 3D).

Next, we analyzed the spatiotemporal distribution of cell death-related proteins following ischemic stroke. Apoptosis can be triggered by intracellular (caspase-independent) and extracellular (caspase-dependent) stimuli. The intracellular pathway relies on the mitochondrial outer membrane permeabilization, resulting in the release of a number of proapoptotic proteins into the cytosol, including AIF. In the extracellular pathway, binding of ligands to a death receptor leads to the activation of caspase-8, which can directly activate caspase-3. FAS-associated factor 1 (FAF1) enhances FAS-mediated apoptosis via the interaction with caspase-8 and FAS-associated death domain protein [12,13]. Activation of caspase-3 results in mitochondrial membrane permeabilization, chromatin condensation, DNA fragmentation, and eventually cell death [1,2,3].

Activated caspase-3 was minimally detected in the sham group, but egan to increase from 0 h reperfusion in the core regions of the striatum and cortex (Fig. 4A). The TUNEL-positive cells were not detected in the sham group but appeared in the striatum and cortex at 0 h (Fig. 4C). Quantitative analyses indicated that both activated caspase-3- and TUNEL-positive cells were gradually increased, peaked on day 1, and then decreased from day 7 onwards (Fig. 4C and D). The expression of these two death markers eventually decreased to the levels of sham group at 1 month.

Similarly, AIF immunoreactivity was not detectable in the sham group, but first detected at 0~2 h only in the striatum (Fig. 5A). The AIF immunoreactivity significantly increased between day 1 and 3 in both the striatum and cortex. Such immunoreactivity decreased from day 7 and no significant immunoreactivity was found in the core region at 1 month. By contrast, AIF immunoreactivity was mainly observed in the cytoplasm in the penumbra from day 1 after reperfusion and was observed in the astroglia-enriched region at 1 month (Fig. 5A).

By comparison, FAF-1 immunoreactivity was detected in the cytoplasm in the sham group but immediately found in condensed cells/nuclei (arrowheads in Fig. 5B) in the striatum from 0 h. FAF-1 immunoreactivity became diffused to the nuclei at very early time points at 0~2 h after the reperfusion in the cortex and then remained in the condensed nuclei (arrows in Fig. 5B). In the penumbra, FAF-1 was localized to the cytoplasm in most cells, while nuclear FAF-1 was detected in some cells (arrows in Fig. 5B). The data indicate that damage to the brain can be evaluated based on the appearance of immunoreactivity against cell death-related molecules in animal models of stroke.

Numerous studies have shown histological lesions with MR images, however, most are limited to specific time and regions. Additionally, there have been no systematic studies on newly discovered cell death-related factors with respect to the images of whole brain lesions. Establishing the spatial and temporal distribution of neuronal necrosis should provide useful guidelines for time limits during which therapeutic intervention may be efficacious.

Ischemic stroke triggers a cascade of events leading to rapid neuronal damage and death [1,2]. At very early time points, such as immediately after 2-h of occlusion (0 h reperfusion), when the infarct is not evident in TTC-stained slices, neuronal degeneration proven by the presence of pyknotic nuclei starts in the striatum (Fig. 1A and B). The infarct progresses to the dorsolateral cortex overlying the striatum at 6 h. Our results are consistent with previous findings that striatal infarction is mostly necrotic and occurs rapidly owing to the nature of lenticulostriate artery, i.e., the lack of collateral circulation, whereas cortical infarction is slightly delayed due to the collateral circulation [7,8].

Activated caspase-3 and TUNEL-positive cells were significantly increased in both the striatum and cortex from an early time point (0 h reperfusion). By comparison, elevation of AIF- and nuclear FAF1-positive cells was detected only in the striatum at 0 h and then in the cortex in the delayed time period. AIF is generally believed to be present in the mitochondria of virtually every type of mammalian cells tested [14,15], and AIF-induced cell death processes are known to be associated with its translocation from the mitochondria to nuclei [16]. Compared to such in vitro studies, immunohistochemical studies on the AIF expression in the brain have been limited. AIF is barely detectable in the normal brain but its expression is dramatically increased in the nuclei in the injured brain [17]. In contrast to such immunohistochemical data, western analysis has revealed the presence of AIF in the cytosolic fraction in the normal brain and its nuclear localization after brain injury [18]. We also could not detect the AIF in the sham group with the antibody that could recognize AIF in the cytoplasm of HeLa cells (Fig. 5A) (Supplementary Fig. 1). Thus, the apparent absence of AIF immunoreactivity in the sham group suggests that AIF in the cytosolic/mitochondrial compartment is masked in the normal brain or was inaccessible under our experimental conditions. FAF1 is also known to be localized in the mitochondrial compartment in the normal condition but it translocates into the nucleus owing to oxidative stress, where it binds to Poly [ADP-ribose] polymerase 1 (PAPR1) to positively regulate the enzymatic activity of the PARP1 both at the cellular level and in a mouse model of PD [19]. Unlike AIF, FAF1 is detectable in the cytoplasm in the sham group but it translocates to the nuclear compartment at 0 h in the infarct core (arrows) and pyknotic nuclei (arrowheads) (Fig. 5B). The subcellular location of FAF1 can be a useful tool for assessing the severity of early cell death in animal stroke models. Taken together, the emergence of immunoreactivity for cell death-related molecules precedes morphological changes in Iba1-positive microglia observed at 6 h. The results are consistent with the previous report that activated caspase-8 and caspase-3/7 trigger microglia activation [20].

The only causal therapy for ischemic stroke is endovascular recanalization of the occluded vessels and reperfusion of ischemic brain parenchyma using thrombolysis agents and thrombectomy devices [21]. Even if cerebral blood flow resumes quickly enough to prevent subsequent cell death, a large population of initially surviving neurons may die within the first few hours after reperfusion. Based on the understanding of pathophysiological changes with respect to the post-ischemic time, antiapoptotic drugs may be beneficial in prohibiting the propagation of apoptosis if given early enough, and administration of anti-inflammatory drugs may be essential to block the secondary inflammatory damages caused by earlier cell death.

Stem cell-based approaches are promising potential therapies to restore brain function after ischemic stroke. Stem cells could exert their neuroprotective effects through anti-inflammation, anti-apoptosis, promotion of angiogenesis and neurogenesis, and formation of new neuronal circuitry [22]. Many laboratories including ours have shown that stem cells can effectively treat stroke via anti-inflammatory functions if given at postischemic day 3 [23]. Our study indicates the anti-inflammatory function of stem cells is effective on day 3 when circulating CD68 + cells are heavily infiltrated into the ischemic nucleus (Fig. 3). Although clinical and preclinical studies on ischemic stroke have produced promising results, several questions remain regarding the possible mechanisms of action of the stem cells and the optimal treatment protocol including the administration route and timing. These parameters may vary with respect to the transplantable cell types [24,25] and the pathogenic microenvironment in the ischemic brain [26,27]. With the rapid development of stem cell therapy, the Stem Cells as an Emerging Paradigm in Stroke (STEPS) committees have set out guidelines focusing on preclinical studies that are considered important parts of a development program to support clinical testing on cell therapies [28]. In STEP3, updated in 2014, the guideline for clinical studies on stem cell therapies for chronic stroke was added [29]. For the treatment of chronic stroke, STEPS3 suggested that the efficacy of stem cell treatment should be proven in animals for at least one month after ischemic injury. From the perspective that the microenvironment of the brain at the chronic stage is destructive, stem cell therapy should at least aim to improve the microenvironment of the brain at the chronic stage. Our results provide information on the spectral changes in the ischemic brain from disease onset to the chronic phase.