To study stroke and potential treatments, rodent models have long been used as one of the important methods. The volume of cerebral infarct can reflect the severity of stroke and correspond to neurological dysfunction. Thus, infarct volume is part of the preclinical study that can help determine the positive impact of any therapy on stroke. Several approaches are available for testing how badly brain damage has been done in experimental stroke models, which are also useful for measuring therapeutic efficacy and comparing studies.

Ace Therapeutics provides reliable infarct volume measurement services to quantify cerebral infarct size in animal models of stroke. We quantitate infarct volume non-invasively using histologically stained brain sections (e.g. hematoxylin and eosin, cresyl violet or 2,3,4-triphenyl tetrazolium chloride (TTC)) or using T2-weighted MRI.

Our infarct volume measurement outcomes provide an objective, quantitative assessment of brain damage and serve as an experimental endpoint or measurement in studies using stroke animals.

Related Services

• Cerebral Infarct Size Measurement Service in Animal Models of Stroke

2,3,5-triphenyltetrazolium hydrochloride (TTC) Staining

TTC is a colorless water-soluble dye that is reduced by the mitochondrial enzyme succinate dehydrogenase of living cells into a water-insoluble, light sensitive compound (formazan) that turns healthy/normal tissue deep red. In contrast, damaged/dead tissue remains white showing the absence of living cells and thereby indicating the infarct region. TTC staining is one of the most common methods widely used to measure the volume of cerebral infarcts in rodent stroke models.

The advantages of TTC staining are that it is inexpensive, simple, and accurate, and can be easily accomplished without complicated equipment and techniques. Most importantly, TTC staining accurately depicts cerebral infarcts and quickly shows infarcts and semi-dark areas. On the other hand, TTC staining has several disadvantages. It is a single-purpose stain used only for macroscopic measurements of cerebral infarction. The time window for TTC staining is relatively narrow.

Fig.1. Representative picture of TTC staining of ischemic rat brain. (Zhang, et al., 2012)

Cresyl Violet (CV) Staining

CV staining highlights early neuronal changes in areas of infarction. Initially, ischemia causes micro vacuolation in the cytoplasm, which corresponds with electron microscopy findings such as swollen mitochondria and dilated endoplasmic reticulum. In the infarct area, there are fewer intact cells compared to normal brain tissue. Consequently, CV staining results in dark blue coloration in healthy regions and light blue in the infarcted areas. Many well-constructed comparative studies have shown a significant correlation between TTC staining and hematoxylin-eosin staining for the quantification of the cerebral infarct.

In contrast, CV staining can be cumbersome due to the requirement for thin sections that must be slide mounted, as well as the need for cryoprotection or paraffin embedding. The process involves time-consuming steps, such as passing sections through graded alcohols and clearing agents.

Fig.2. Representative coronal sections of mouse brain stained with cresyl violet after 72 hr post-reperfusion. (Rousselet, et al., 2012)

Histological Staining

Various histological staining methods are used to visualize ischemic brain regions. Nissl staining, using an aniline stain to label extranuclear RNA granules which mainly localize the soma of neurons, is a preferable staining method for microscopic evaluation of ischemic tissue (triangular shaping, exhibiting dark staining due to condensation of cytoplasm and karyoplasm). Hematoxylin and eosin (HE) staining remains the most common method and is often considered the "golden standard" for measuring infarct volume, despite some artifacts, such as the appearance of 'dark neurons'.

However, histological processing can introduce changes in brain morphology (swelling/shrinkage) that affects the accuracy of lesion size measurements. Additionally, histological staining is manual-labor-intensive and can be subject to variability in staining intensity and interpretation leading to errors in lesion size quantification. The choice of the staining methods or the criteria used to define the lesion border can make it difficult to compare ischemic areas across different studies.

Fig.3. Ischemic infarction evaluated by Nissl staining. (Li, et al., 2014)

Magnetic Resonance Imaging (MRI)

MRI has become a promising imaging modality in the preclinical lab thanks to the introduction of small animal scanners with high resolution that can quantify lesion volume in stroke with precision and accuracy. Moreover, MRI is also a very helpful means to translate preclinical research because it is so ubiquitous in the clinical realm. MRI-measured lesion volumes provide an objective and quantitative measure of stroke severity. Diffusion-weighted imaging (DWI) is used to detect ischemia in the acute phase, and T2-weighted fluid-attenuated inversion recovery (FLAIR) imaging is used to visualize vasogenic edema and infarction in the subacute and chronic phases. Standards for lesion measurement have been developed and acute lesion development has been extensively studied in a variety of settings. However, accessibility of MRI in preclinical studies is still limited due to the high cost of equipment and maintenance.

Fig.4. T2-weighted structural MRI is used to measure the size of the infarct and brain swelling after stroke. (Wayman, et al., 2016)

1. Zhang, F., & Chen, J. (2012). Infarct measurement in focal cerebral ischemia: TTC staining. Animal Models of Acute Neurological Injuries II: Injury and Mechanistic Assessments, Volume 2, 93-98.
2. Rousselet, E., et al. (2012). Mouse model of intraluminal MCAO: cerebral infarct evaluation by cresyl violet staining. Journal of visualized experiments: JoVE, (69), 4038.
3. Li, H., et al. (2014). Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice. BMC neuroscience, 15, 1-13.
4. Wayman, C., et al. (2016). Performing permanent distal middle cerebral with common carotid artery occlusion in aged rats to study cortical ischemia with sustained disability. Journal of visualized experiments: JoVE, (108), 53106.