Comparison of the marker effects of two different fluorescent dyes in labeling endogenous neural stem cells in the central nervous system / 解放军医学杂志

ABSTRACT

Objective To observe the marker effects of two different fluorescent dyes, DIL and DAPI, in labeling endogenous neural stem cells (ENSCs) in rat central nervous system. Methods Thirty-six Sprague-Dawley rats were randomized into staining groups, comprising DIL group and DAPI group, and the corresponding control groups, including DMSO group for DIL group and PBS group for DAPI group. 0.2% DIL 10μl or 10μg/ml DAPI 10μl was stereotactically injected into the lateral ventricle of rats of DIL group or DAPI group, while DMSO or PBS 10μl was introduced into that of DMSO group or PBS group. Neurological severity score (NSS) was determined 2 hours and 24 hours respectively after the operation. Rats were sacrificed at day 1, 3, 7 after the injection. Serial coronal sections of the brain and spinal cord were carried out on a cryostat, and then they were observed under a confocal microscope. The fluorescence intensity of the targeted area, which highlighted by labeled ependymal cells, in the brain and spinal cord of cervical vertebrae, thoracic vertebrae and lumbar vertebrae were semi-quantified. Fluorescence intensity of each section was measured in triplicate, and a mean value was obtained. Statistical analysis was performed on 3 data sets, randomly selected from sections of brain and spinal cord obtained at day 1, 3, 7. Results Two hours after DIL injection, the rats showed no evident neurological defect. NSS value was very low, and there was no significant difference compared with the DMSO group (P>0.05). Twenty-four hours later, normal neurological function recovered in all the rats. Red fluorescence could be seen in the cytoplasm of ependymal cells in the lateral ventricle and each spinal cord segment at day 1 after the DIL injection, and it did not disappear until the 7th day. Nuclei of DAPI-labeled lateral ventricle cells were blue, with clear nuclear morphology. Choroid plexus cells of the ventricle were also labeled. However, there was no blue fluorescence in the medulla oblongata or any segment ofthe spinal cord. The picture at day 3 and day 7 was similar to that of day 1. No significant difference was found between fluorescence intensity in DIL or DAPI stained cells(P>0.05) at any time point. Conclusions DIL may serve as a marker of the cytoplasm of ependymal cells in the brain ventricle and spinal central canal. DAPI, which is often used in the nuclear staining, can label the ENSCs in the brain ventricle. Intraventricular injection of fluorescent dye is a relatively safety procedure.