An objective procedure for ischemic area evaluation of the stroke intraluminal thread model in the mouse and rat
Introduction
The intraluminal thread middle cerebral artery occlusion model is one of the models established to mimic aspects of the pathophysiology and progression of stroke in humans. This model permits the induction of both permanent as well as transient ischemia (Koizumi et al., 1986, Longa et al., 1989) and has the benefit of using small animals so that a large number of studies can be performed under controlled conditions. However, this model has a high degree of variability. The consistency, reproducibility and location of ischemic injuries can, and often do, differ quite markedly (Schmid-Elsaesser et al., 1998) and minor technical changes significantly affect the outcomes. Such variability can be further exacerbated by the method used to measure the lesions. For this reason, different staining techniques and methods for imaging brain sections have been used (Goldlust et al., 1996, Vogel et al., 1999, Yang et al., 1998). One such staining technique uses 2,3,5-triphenyltetrazolium chloride (2,3,5-TTC) for the identification of areas of necrosis and infarct (Vogel et al., 1999).
We have refined the intraluminal thread model by using sham-operated animals as baseline controls and by using imaging techniques that decrease the subjectivity of data analysis. Sham-operated animals were used to evaluate normal white matter that does not stain with TTC. Using brain sections from sham-operated animals and Image Pro Plus™ with color segmentation, we developed an objective method to assess ischemic damage. Major benefits of our approach include: objectivity of the analysis of the ischemic injury, cost-containment because of the use of readily available software and removal of the normal subcortical white matter from the analysis of the injured brain.
Section snippets
Chemicals
All reagents were chemical grade and purchased from Sigma Chemical Co. (St. Louis, MO) or through VWR (Bridgeport, NJ).
Animals
All animal studies were conducted in a facility accredited by the American Association for the Accreditation of Laboratory Animal Care. Male Sprague–Dawley rats (250–300 g) and male C57B1/6 mice (22–25 g) (Charles River Laboratory, Wilmington, MA) were used.
Mouse and rat cerebral ischemia models
Animals were anesthetized with 1–2% isoflurane in N2O and O2. Body temperatures were maintained at 37 °C using a
Brain scans
Fig. 1A–D includes the entire scanned sections from one representative normal rat brain. The anterior and posterior sections are not used in the assessment of brain tissue, since these sections are variable (see Section 2). Sections E–H are from a representative permanent occlusion ischemic rat brain at 24 h, with the lesion clearly visible. A and E are the raw scans, B and F are the optimized scans and C and D are the red while D and H are the green channels.
Fig. 2 shows representative scanned
Discussion
A number of animal models have been developed over the last two decades or so, but none have been shown to be predictive for man, and probably will not be until a drug used in that specific model demonstrates neuroprotection in man following stroke (Hunter et al., 1995). Animal models of cerebral ischemia mimic less than 25% of all strokes (Small and Buchan, 2000). However, some of the lesions observed in the ischemia models are similar to those observed after stroke in humans (Hunter et al.,
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