DEVELOPMENT OF ENDOGENOUS BETA-GALACTOSIDASE AND AUTOFLUORESCENCE IN RAT-BRAIN MICROVESSELS - IMPLICATIONS FOR CELL TRACKING AND GENE-TRANSFER STUDIES

Cell transplantation is commonly used in studies of CNS development, t umor 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 beta-galactosidase (beta-Gal) activity. Our interest in the CNS vascul ature led us to examine vascular autofluorescence and beta-Gal activit y in postnatal and adult rat brains. Brains were perfusion-fixed (3.7% paraformaldehyde), cryoprotected, and cryostat-sectioned (12 mu m). A utofluorescent vessel pro-files were quantitated in sections using rho damine filter sets and beta-Gal-positive vessels were quantitated unde r bright-field after incubation of sections with X-Gal chromogenic sub strate for 1-18 hr at 37 degrees C. Multifocal vessel autofluorescence appeared in postnatal Day (PND) 18 Lewis rats (0.6 +/- 0.4 vessels/fi eld) and increased tenfold in adults (6.8 +/- 0.3/field). The numbers of beta-Gal-positive vessels in PND 18 and adult sections incubated wi th X-Gal for 18 hr were 21.1 +/- 1.7 and 119 +/- 17, respectively. Hos t beta-Gal staining was similar to that produced by implanted endothel ial cells expressing the bacterial lacZ reporter gene. Reducing incuba tion times in X-Gal to less than 4 hr eliminated endogenous staining a nd retained lacZ-specific staining. The presence of vascular autofluor escence and endogenous beta-Gal activity must be considered when eithe r fluorescence- or lacZ-dependent cell markers are used in rat brain.