Mannitol-facilitated perfusion staining with 2,3,5-triphenyltetrazolium chloride (TTC) for detection of experimental cerebral infarction and biochemical analysis

https://doi.org/10.1016/j.jneumeth.2011.09.029Get rights and content

Abstract

A simple method to quantify cerebral infarction has great value for mechanistic and therapeutic studies in experimental stroke research. Immersion staining of unfixed brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) is a popular method to determine cerebral infarction in preclinical studies. However, it is often difficult to apply immersion TTC-labeling to severely injured or soft newborn brains in rodents. Here we report an in vivo TTC perfusion-labeling method based on osmotic opening of blood–brain-barrier with mannitol-pretreatment. This new method delineates cortical infarction correlated with the boundary of morphological cell injury, differentiates the induction or subcellular redistribution of apoptosis-related factors between viable and damaged areas, and easily determines the size of cerebral infarction in both adult and newborn mice. Using this method, we confirmed that administration of lipopolysaccharide 72 h before hypoxia–ischemia increases the damage in neonatal mouse brains, in contrast to its effect of protective preconditioning in adults. These results demonstrate a fast and inexpensive method that simplifies the task of quantifying cerebral infarction in small or severely injured brains and assists biochemical analysis of experimental cerebral ischemia.

Highlights

Osmotic opening of blood–brain-barrier allows in vivo TTC labeling of the brain. ► In vivo TTC-labeling quantifies infarction in badly injured or small newborn brains. ► In vivo TTC-labeling supports biochemical analysis of the ischemic penumbra area.

Introduction

Immersion staining of fresh brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) is a simple and popular method for detecting infarction in experimental stroke models (Bederson et al., 1986). TTC, a colorless water-soluble dye, is reduced to a deep red, water-insoluble compound (formazan) predominantly in the mitochondria of living cells, hence distinguishing between viable and infarcted brain tissue after stroke. However, immersion TTC-staining has its limitations because it is often difficult to section unfixed, edematous brains after severe ischemic injury, especially in newborn rodents. While perfusion staining of rodent brains by intracardiac injection of TTC solution was reported, we have found this method to be very inefficient (see Section 3), and the previous TTC perfusion-labeling method was rarely adopted in the literature (Isayama et al., 1991). The lack of a reliable in vivo TTC-labeling method has increased the workload of quantifying tissue damage in experimental stroke, and uncertainty in distinguishing the core-versus-peri infarct areas for biochemical analysis.

Interestingly, although intracardiac injection of TTC hardly labels the brain, it stains the heart efficiently and has been used widely to detect experimental myocardial infarction. This disparity suggests to us that the entry of TTC dye to central nervous system may be hindered by the blood–brain-barrier (BBB). To test this hypothesis, we performed TTC perfusion-labeling after osmotic BBB disruption with mannitol (Rapoport and Thompson, 1973, Rapoport, 2000), and found a greatly enhanced brain-staining capacity. Here we report optimization of the TTC perfusion-labeling method that demarcates infarction in both adult and newborn mouse brains. Further, we show that the in vivo TTC-labeling method is compatible with biochemical analysis and distinguish between viable and infarcted tissue. Finally, using this method, we showed that pre-exposure to a bacterial endotoxin lipopolysaccharide (LPS) 72 h before hypoxia–ischemia amplifies brain damage in newborn mice, in contrast to its effect of protective preconditioning in adults (Tasaki et al., 1997, Rosenzweig et al., 2004, Eklind et al., 2005). Additional applications of this new vitality-detection method are also discussed.

Section snippets

Animal surgery

For adult cerebral hypoxia–ischemia (HI), eight-to-twelve week-old male CD1 (Charles River, Wilmington, MA) and Thy1-YFP mice (Jackson Laboratories Stock no. 003782, Bar Harbor, ME) were challenged by transient cerebral HI, performed as described previously with minor modifications (Adhami et al., 2006, Shereen et al., 2011). Briefly, animals were anesthetized by intraperitoneal (IP) injection of avertin, and the right common carotid artery was ligated by two releasable (Mule) knots of 4-O silt

Optimization of TTC perfusion-labeling by mannitol-induced BBB disruption

First, we compared the ability of intracardiac versus intra-carotid artery perfusion of 2% TTC solution to stain the brain (Isayama et al., 1991, Dettmers et al., 1994). Whereas intra-artery (IA) injection of TTC worked better than intracardiac perfusion, neither method provided strong staining of the brain, despite intense labeling of the liver (Fig. 1A) and heart (data not shown). In contrast, following intraperitoneal (IP) injection of 1.4 M mannitol (250 mg/g body weight) that allowed the

Osmotic opening of BBB facilitates TTC perfusion-labeling

The ability to quickly determine the location and extent of infarction after experimental cerebral ischemia is important for the investigation of underlying mechanisms and assessment of new interventions. While immersion TTC-staining is a very useful method to quantify cerebral infarction, it is less applicable to severely injured, edematous brains or smaller newborn brains in rodents. The alternative methods, including comparison of neural injury scores or quantification of the area devoid of

Conclusion

In conclusion, our results demonstrated a mannitol-facilitated TTC perfusion-labeling method that is a useful alternative to the ex vivo immersion-labeling method. The in vivo TTC-labeling method is simple, inexpensive, and particular useful for measuring injury in severely damaged or small newborn brains. This new vitality-detection method not only simplifies the task of quantifying infarction, but also supports biochemical analysis of the core- and peri-infarct areas in experimental stroke.

Acknowledgements

This work was supported by National Institute of Health Grant NS 074559 (to C.-Y.K) and an American Heart Association fellowship (to D.Y.).

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