[An improved quantitative method for evaluation of ischemic injury and neuroprotection in mouse brain slices]

To establish a simpler and more accurate method for evaluating in vitro ischemic injury and neuroprotective effects of drugs through improving experimental instrument and quantitative index in mouse brain slices.

An incubation instrument was developed and its application tested. 2,3,5-triphenyltetrazolium chloride (TTC) was used as a substrate to biosynthesize formazan standard in mouse brain slices, and formazan was isolated, purified and identified. Ischemic injury of mouse brain slices was induced by oxygen/glucose deprivation (OGD), the produced formazan from TTC in the cortex and striatum was measured at 490 nm spectrophotometrically. Edaravone and ONO-1078 were added into the incubation medium to observe their neuroprotective effects.

The incubation instrument worked well for incubating brain slices and obtaining stable results efficiently. Standard formazan was biosynthesized and purified with a purity of 99.3%, and showed a linear range of 0.05 - 1 mg/ml in absorbance at 490 nm (r=0.9997). OGD decreased formazan production in the cortex and striatum in a duration-dependent manner. Edaravone (0.01 to 1 micromol/L) recovered OGD-induced decrease of formazan production, but ONO-1078 showed no effect.

The incubation instrument and quantitative measurement of formazan developed in this study are efficient,accurate and simple for evaluating ischemic injury and neuroprotection,which can be used in screening of neuroprotective drugs in vitro.