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Literature Review | Mouse Stroke Model: High-Resolution Micro-CT Imaging—From Infarct Characterization to Automated Region Segmentation

Introduction:

Stroke, also known as “apoplexy” or “cerebrovascular accident,” is an acute cerebrovascular disorder characterized by brain tissue damage resulting from the sudden rupture of a cerebral blood vessel or from vascular occlusion that prevents blood from reaching the brain. It encompasses both ischemic stroke and hemorrhagic stroke.

Ischemic stroke is a leading cause of death and disability, and the second most common cause of dementia.

The most commonly used model of cerebral stroke is the transient middle cerebral artery occlusion (tMCAO) model. This model can be employed to investigate the mechanisms and outcomes of ischemic injury at varying severities. The tMCAO model induces a permanent lesion in the striatum—the infarct core—along with a penumbra in the surrounding cortical regions. The penumbra is an area of reduced blood flow that, despite diminished perfusion, retains structural integrity during stroke.

In recent years, the ischemic penumbra has attracted increasing attention, and this region has become the focal point of many current strategies for treating stroke. Micro-CT scanning and analysis of animal models can provide a comprehensive and detailed assessment of pathological changes within the brain.

 

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In the early stages of stroke, the penumbra represents the largest portion of the infarct volume; without reperfusion, it will progress to an irreversible cerebral infarction. Therefore, precise localization and temporal expansion of this highly dynamic region—also known as the peri-infarct zone—and the delineation of the core infarct area are of critical importance. A detailed characterization of white‑matter–level brain injury is considered a key metric for understanding the pathophysiology of stroke.

Interpreting the Literature Source

 

Post-stroke characterization in rodent models relies on histological analysis, including contrast-enhanced staining of brain sections and immunohistochemistry.

However, brain sectioning can cause severe tissue damage and distortion, as well as the loss of some samples and the disruption of the brain’s native 3D architecture. Conventional histological analysis cannot provide this information; only 3D imaging techniques can. This is where micro-CT comes into play, offering high spatial resolution, flexibility, and low cost for preclinical research.

The main drawback of micro-CT is the low X-ray attenuation of soft tissues. This issue can be overcome by employing contrast agents—such as osmium tetroxide (OsO4), iodine, phosphomolybdic acid (PMA), and phosphotungstic acid (PTA)—to enhance the visibility of soft tissues.

 

Materials and Modeling Methods

Male and female wild-type C57BL/6 mice aged 3 to 12 months were used. A tMCAO procedure was performed: the external carotid artery and its branches were occluded, and the pterygopalatine artery was also ligated to interrupt extracranial collateral circulation. A suture was inserted through the external carotid artery, advanced into the internal carotid artery and further into the anterior cerebral artery, thereby mechanically blocking blood flow at the origin of the middle cerebral artery.

 

Experimental Results

High-resolution micro-CT imaging enables the precise identification of anatomical regions in the brain.

Different soft-tissue stains were applied to the entire mouse brain, and acquiring the image dataset took 3 to 5 hours.

Among all the compounds tested, PTA and PMA failed to stain the entire brain, suggesting they do not fully penetrate the tissue (Supplementary Fig. 1B,C).

Taking iohexol as an example, although it can penetrate the entire sample, it does not provide good contrast between different brain structures (Supplementary Figure 1A). In contrast, brain sections stained with osmium tetroxide (Figure 1B) and inorganic iodine (Figure 3) exhibit high-contrast imaging of brain structures, with a resolution of 3.5–4 μm voxels, enabling microscale reconstruction of whole-brain three-dimensional volumes (Figures 1a and b—coronal, sagittal, and axial views).

Figure 1

 

Two staining methods—osmium tetroxide and inorganic iodine—were clearly distinguishable under Micro-CT imaging, as observed in the cortex (Ctx), striatum (CPu), hypothalamus, thalamus (TH), and hippocampus (HP) (Fig. 1B and Supplementary Fig. 3). In terms of toxicity, inorganic iodine also exhibited significant advantages over osmium tetroxide (Table 1).

 

Table 1: Comparison of the distinct characteristics of whole-brain contrast enhancement using osmium tetroxide versus inorganic iodine staining.

High-resolution micro-CT enables the visualization of stroke‑related lesions in the mouse brain. Following ischemic injury, the brains are stained with osmium tetroxide or an iodine‑based contrast agent. Micro-CT images are acquired, and the resulting data are reconstructed into three-dimensional projections using X‑ray tomography.

Despite differences in image contrast, both osmium tetroxide– and inorganic iodine–stained brains exhibit clear signs of ischemic injury (Fig. 1C, 2A—osmium—and Supplementary Fig. 3, iodine), including cerebral edema and neuronal degeneration within the infarcted regions.

At the lesion site, 24 hours after reperfusion, distinct differentiation of the core and penumbra was observed (Fig. 2A–C, Suppl).

Iodine‑stained brains can be subjected to further histological processing, preserving the structural components recognized by the antibodies used in the immunofluorescence protocol (Supplementary Figure 2b). Due to the dehydration step in the iodine‑staining procedure, some brain atrophy was observed. The final brain volume was calculated to be one third of the initial brain volume (Supplementary Figure 2A).

In contrast, although brains stained with osmium tetroxide do not exhibit brain atrophy, the staining process induces tissue mineralization, further precluding their use in immunofluorescence.

Figure 2

 

 

 

Characterization of Cerebral Infarction Lesions in Mice 

Assessing stroke treatment requires detecting subtle differences in infarct volume. By optimizing contrast‑enhanced Micro‑CT imaging with OsO4 or inorganic iodine, the researchers demonstrated that this approach is suitable for delineating the boundaries of ischemic lesions, including distinguishing the ischemic core from the penumbra as early as 24 hours after reperfusion. It also enables visualization of cerebral edema (Figure 2A and Supplementary Figure 3) and allows for longitudinal monitoring of brain lesions over time, from 3 to 72 hours (Supplementary Figure 5) and up to 7 days (tested but not shown).

Figure 3

 

Figure 5

 

Using the aforementioned method, it was possible to observe that the infarct volume in the cortex and striatum increased over time, along with a corresponding enlargement of the ipsilateral hemisphere due to edema (3–24 hours after reperfusion) (Supplementary Figure 5).

The lesion area was analyzed using a 3D image dataset acquired by micro-CT. These measurements were compared with data obtained from standard triphenyltetrazolium chloride (TTC) staining (Figure 8), as well as with immunostaining for NeuN (a neuronal marker), MAP2 (a neuronal marker), and TUNEL (a late-stage apoptosis/necrosis marker) (Figure 2D).

From the analysis, the lesion area calculated from the virtual slice closely matches the lesion area quantified on the histological section, confirming that the delineated boundary corresponds to the ischemic infarct area (Fig. 2D and Fig. 8C,D; see Fig. 8 below).

Furthermore, Micro-CT imaging revealed small lesions in myelinated fibers following a TIA model in mice.

 

Micro-CT enables precise, manual quantification of brain injury and edema in mice.

Using Bruker®CTAn’s proprietary micro-CT software, the stained regions of both cerebral hemispheres and the ischemic lesion were manually outlined in virtual cross-sections (Figure 5), and the extent of edema (percentage of the contralateral hemisphere) (Figures 4A–C) as well as the corrected lesion volume (as a percentage of total brain volume) (Figures 4D–F) were further calculated (see Materials and Methods).

Figure 4

 

Deep learning has enabled fully automated lesion segmentation in Micro-CT images.

To enhance reproducibility, enable high-throughput quantification, and minimize operational bias, we developed an automated segmentation pipeline for quantifying micro-CT–derived lesions. The results of automated quantitative analysis of iodine‑stained brain tissue in the tMCAO model are presented in Table 2.

Table 2: Comparison of Automated Quantitative Results of tMCAO Brain Iodine Staining Based on Deep Learning Segmentation with Manual Segmentation

These results demonstrate that high-resolution ex vivo micro-CT with contrast enhancement is a major advantage in preclinical stroke research in mice, improving experimental efficiency compared with conventional histological methods.

 

Comparison of Micro-CT Imaging and Histological Staining in Mice

The lesion volume and edema severity derived from micro-CT–based three-dimensional reconstruction datasets were correlated with histological TTC‑based quantification of lesions (Figure 8A). However, TTC staining has limitations: it can only be performed on fresh, thick tissue sections.

Figure 8

 

Table 3: Names and abbreviations of brain regions affected by ischemia

 

Discussion

In rodent stroke models, micro-CT imaging offers significant advantages for detecting and precisely quantifying post‑ischemic brain injury ex vivo, as well as for assessing white matter degeneration. It enables simple, rapid, and non‑invasive three‑dimensional imaging of the entire mouse brain, with high resolution and fine anatomical detail.

Iodine and osmium staining of mouse brain tissue during the ischemic period allows visualization of the temporal evolution of ischemic injury features. The extent of ischemic damage can be deliberately modulated, with a well-defined core region (when present), and correlates positively with the quantitative assessment of total lesion volume obtained by TTC staining.

 

References:

doi: https://doi.org/10.1101/2022.02.02.478782

Baidu entry: Stroke

 

 

Aniphe BioLab offers Micro-CT imaging services (medium resolution), suitable for both ex vivo tissues and live small animals.

Other categories of imaging services include: B‑ultrasound, X‑ray imaging, in vivo imaging system for small animals (IVIS), photoacoustic imaging, photothermal imaging, magnetic resonance imaging, PET/CT scanning, and a conscious‑animal body composition analyzer.

Aniphe BioLab also offers custom disease‑animal model services. Feel free to contact us!

 

 

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