| Quantification of cerebral ischemic infarction has been achieved most commonly by ' gold ' standard methods such as hematoxylin and eosin (HE) staining and 2,3,5-triphenyltetrazolium chloride (TTC) staining . However, HE staining must be performed on fixed tissue. TTC staining can determine total infarct area around 24h after the onset of ischemia, but it is controversial whether this technique is suitable to define brain infarct size when employed within 4-6h and after 36h of ischemia. HE or TTC stained sections can not be used for further investigations such as biochemistry, histology and immunohistochemistry. To develop a rapid, quantitative, novel method for evaluating infarct size in focal cerebral ischemic model, we have evaluated the feasibility of light transmission to measure focal cerebral ischemia in mice. The relationship between cerebral infarct volumes measured by light transmission and neurological scores has been analyzed. We have compared infarct size measured by light transmission with survived neurons in HE stained sections. Furthermore , light transmission together with measurement of neurological score and HE staining has been employed to demonstrate the neuroprotective activity of ONO-1078 { pranlukast , 4-oxo-8-[p-(4-phenylbutyloxy)benzoyl-amino]-2-(tetrazol-5-yl)-4H-l-benzopyran hemihydrate} , a potent leukotriene receptor antagonist, in a model of focal cerebral ischemia in mice.MethodsSixty male NIH mice were divided into ischemic group (n=40) and sham group (n=20). Persistent focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO), and in sham group mice, common carotid artery was exposed without occlusion. Bederson's neurological scores, climbing board and hanging test were performed 24h after surgery. Then, the animals were decapitated and the brains were removed and coronally dissected into 6 sections with 1mm thickness. Using a stereomicroscope, each section was illuminated with a halogen lamp and computerized images were stored. The brain sections were then stained for 30min with 0.5% 2,3,5-triphenylterzolim chloride (TTC) at 37癈 and images were stored. Using an image analyzer (AnalyPowerl.O), the infarct volumes obtained by light transmittance and TTC staining were calculated. Integrated gray scales of sections of both hemispheres were calculated by Photoshop 5.0. Coronal cryostat sections Sum thick were prepared from the fifth brain section. After HE staining, the brain section was observed under light microscope and the image (X600) was stored in the computer. Amounts of survived neurons in cortex and subcotex were counted.In another series, ONO-1078 (O.lmg-kg"1, n=15) or normal saline (n=14) were injected ip once daily for 3 days and at Ih before MCAO to evaluate the neuroprotective effect of ONO-1078. ResultsThe normal tissues of brain sections essentially translucent and the ischemic areas were visible to grow dark and gray scales increase in light transmittance experiments. TTC staining of the normal tissue in brain sections was wine-colored, and the ischemic areas whitened with gray scales decreased. A close correlation existed between cerebral infarct volumes measured by light transmission and TTC staining (r = 0.809, P < 0.001). The mean gray scales measured by both techniques of the hemispheres as well as the cortex,subcortex and hippocampus in ischemic hemispheres were significantly different fromthose of non-ischemic hemispheres and of control hemispheres (P < 0.001). There were no significant differences between the two hemispheres of control mice and between hemispheres of control mice and non-ischemic hemispheres of the MCAO mice. The neurological scores measured by integrated graded approach in ischemic mice were higher than in control mice (P < 0.001). The cerebral infarct volumes measured by light transmission were closely correlated with the neurological scores measured by integrated graded approach (rs=0.694, P < 0.001). The survived neurons in HE stained sections of cortex and subcotex in ischemic group were...
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