Development of endogenous β-galactosidase and autofluorescence in rat brain microvessels: Implications for cell tracking and gene transfer studies

Abstract

Cell transplantation is commonly used in studies of CNS development, tumor biology, and gene therapy. Fluorescent dyes and the E. coli lacZ reporter gene are used to identify transplanted cells in host tissues. The usefulness of these methods depends on host autofluorescence and β-galactosidase (β- Gal) activity. Our interest in the CNS vasculature led us to examine vascular autofluorescence and β-Gal activity in postnatal and adult rat brains. Brains were perfusion-fixed (3.7% paraformaldehyde), cryoprotected, and cryostat-sectioned (12 μm). Autofluorescent vessel profiles were quantitated in sections using rhodamine filter sets and β-Gal-positive vessels were quantitated under bright-field after incubation of sections with X-Gal chromogenic substrate for 1-18 hr at 37°C. Multifocal vessel autofluorescence appeared in postnatal Day (PND) 18 Lewis rats (0.6 ± 0.4 vessels/field) and increased tenfold in adults (6.8 ± 0.3/field). The numbers of β-Gal-positive vessels in PND 18 and adult sections incubated with X-Gal for 18 hr were 21.1 ± 1.7 and 119 ± 17, respectively. Host β- Gal staining was similar to that produced by implanted endothelial cells expressing the bacterial lacZ reporter gene. Reducing incubation times in X- Gal to less than 4 hr eliminated endogenous staining and retained lacZ- specific staining. The presence of vascular autofluorescence and endogenous β-Gal activity must be considered when either fluorescence- or lacZ- dependent cell markers are used in rat brain.