1Department of Anesthesiology, The First Clinical College, China Medical University, Shenyang 110001, China
2Jiangsu Province Key Laboratory of Anesthesiology, 3Jiangsu Key Laboratory of Brain Disease Bioinformation, 4Nursing School, Xuzhou Medical College, Xuzhou 221002, China
*These authors contributed equally to this work.

Abstract 

It has been reported that distal cerebrospinal fluid-contacting neurons (dCSF-CNs) can be detected by immunohistochemical assay using cholera toxin subunit B-conjugated horseradish peroxidase (CBHRP). In the present study, another two methods with CB alone or CB-conjugated FITC (CB-FITC) were used, and the results from the three methods were compared. Adult Sprague-Dawley rats were randomly divided into three groups with CB-HRP, CB or CB-FITC. Tracers were diluted to 30% in artificial cerebrospinal fluid and injected separately (in a volume of 3 μL) into the lateral ventricle. Animals from the CB-HRP and CB groups were perfused 48 h after surgery while animals from the CB-FITC group were perfused 1, 3, 6, 12, 24 or 48 h after surgery. The brain was sectioned (40 μm) for immunofluorescence and five sections with positive neurons were selected from each rat for neuron counting. Three clusters of positive neurons in a ‘Y-like’ distribution were detected ventral to the cerebral aqueduct of rats from the three groups. No significant difference was observed among the quantitative data. In the CB-FITC group, stable staining was detected even at 6 h after injection. Taken together, lateral ventricle injection of CB/CB-FITC is a useful method for labeling dCSFCNs in rats. The CB-FITC method makes dCSF-CNs labeling much simpler and more convenient.

Keywords

distal cerebrospinal fluid-contacting neurons; cholera toxin subunit B-conjugated horseradish peroxidase; cholera toxin subunit B; cholera toxin subunit B-conjugated FITC; label

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