Comparison of the chronic toxicity of piroxantrone, losoxantrone and doxorubicin in spontaneously hypertensive rats

We have determined the time course, the spatial spread in brain tissue, and the intracellular distribution of biotin- and fluorescein-labeled phosphorothioate oligodeoxynucleotides (ODNs) following single injections into the rat striatum or the lateral ventricle. These time and space parameters were correlated with the ability of c-fos phosphorothioate antisense ODNs to suppress the induction of Fos protein by cocaine. A rapid and dose-dependent tissue penetration of labeled ODNs was observed following either intrastriatal or intraventricular injections of a constant sample volume. Inspection of tissue sections by confocal microscopy uncovered a distinct change in the intracellular disposition of labeled ODNs during the 24 h post-injection period. At 1, 6 and 12 h, the vast majority of the fluorescent signal was confined to the interstitial spaces throughout the zone penetrated by ODNs. Neuronal nuclei displayed faint labeling along the outer portion of the nucleus at 1 and 6 h post-injection. At these time-points, ODNs were not detected in the cytoplasm. By 16 h, ODNs were barely detectable in the extracellular space and absent from neuronal nuclei. Instead, ODNs were seen in large cytoplasmic granules of neurons throughout the tissue zone penetrated by the ODNs. Experiments with intrastriatal injections of antisense ODNs to c-fos mRNA revealed Fos suppression between 3 and 12 h, but not at 16 and 24 h. This combined analysis has revealed that (1) restricted tissue penetration by ODNs limits their antisense effects on protein expression, and (2) depletion of extracellular ODNs and sequestration of c-fos antisense ODNs into large intracellular granules coincides with the loss of their biological activity.