Distribution and toxic effects of intravenously injected epirubicin on the central nervous system of the mouse.

Epirubicin (4'-epi-doxorubicin) is a new anthracycline cytostatic, which was synthesized in an effort to find an agent with an improved therapeutic effect on human malignant tumours combined with reduced myocardial toxicity. Animal experiments have previously shown that the parent drug doxorubicin, besides being a myocardial toxin, may cause nerve cell lesions both in the central and the peripheral nervous system. Intravenously (i.v.) injected doxorubicin passes into regions of the nervous system located outside the blood-brain barrier (BBB); the drug accumulates in the nucleus of neurons and causes cell degeneration. This investigation was performed to establish whether epirubicin, given as a single i.v. injection in the mouse, could enter the central nervous system (CNS) and cause neurotoxic effects. Epirubicin was found to emit a primary orange fluorescence in thin frozen sections. Detectable amounts of epirubicin could not be seen in regions protected by the BBB. The choroid plexus and all the circumventricular brain regions with the exception of the subcommissural organ showed the presence of the drug in the parenchyma and a marked accumulation in cell nuclei. Severe cellular changes were found in these regions by light and electron microscopy and the alterations were most marked in mice with the longest survival period (45 days). The animals also developed a progressive sensory polyneuropathy and appeared lethargic. Epirubicin given as a single i.v. injection in the mouse will thus spread into brain regions lacking a BBB where it produces toxic lesions in the same way as previously reported for the parent compound, doxorubicin.

Degeneration of trigeminal ganglion neurons caused by retrograde axonal transport of doxorubicin.

Selective nerve cell degeneration was induced in the trigeminal ganglion of the mouse by injecting doxorubicin (Adriamycin) around intact sensory nerve terminals of the head. The drug apparently reached the neurons by retrograde axonal transport after its uptake in nerve branches. A direct fluorescence microscopic method revealed that the compound accumulated in the neurons. Electronmicroscopy showed degeneration of these cells, beginning in the nucleolus and the nucleus. Doxorubicin injected around sensory nerve terminals appears to be a useful compound for selective destruction of mouse sensory neurons. Retrograde axonal transport of neurotoxic compounds is probably an important pathogenetic mechanism in certain forms of intoxication which give rise to lesions in the nervous system.

Doxorubicin as a fluorescent nuclear marker in tumors of the human nervous system: a simple and reliable staining technique.

This paper concerns the use of the fluorescent cytostatic compound doxorubicin (adriamycin) as a simple and reliable staining technique for nuclear DNA on tissue sections, isolated cell smears and imprint preparations. This method can be applied successfully to formalin-fixed, paraffin-embedded specimens of human brain tumors. The distribution and concentration of chromatin within each cell nucleus can be assessed with great accuracy due to the distinct orange fluorescence of doxorubicin. Various cell types can be identified by their nuclear chromatin pattern, and signs of cellular malignancy can be revealed by the drug-induced fluorescence. The new method seems to be suitable both for routine neuropathological work and for tumor research. Quantitative studies on the nuclear DNA content can be easily and rapidly performed, since the method does not require a previous extraction of the cellular RNA.

Cytofluorescence localization of propidium iodide injected intravenously into the nervous system of the mouse.

Propidium iodide, like its analogue ethidium bromide, is a compound which can be used as a marker of nucleic acids. This substance emits a red fluorescent light after exposure to UV light and has therefore been used previously as a nuclear stain in immunofluorescence studies and in flow cytometry. The present experiments were carried out to find out if propidium iodide could be traced in sections of the nervous system after i.v. injections. Due to the general toxicity of the compound detectable amounts of propidium iodide could not be obtained by a single i.v. injection. However, multiple injections of small amounts (0.1 mg) over a period from 15 min to 8 h (total dose 0.7-1.0 mg) were tolerated without any signs of adverse effects. In such experiments propidium iodide did not extravasate into the cerebral gray or white matter, i.e., areas of the brain located within the blood-brain barrier (BBB). On the other hand, the compound spread into the choroid plexus, the circumventricular organs, the Gasserian ganglion, and sciatic nerve, i.e., regions located outside the BBB. It had a strong tendency to label the nucleus and the perikaryon of the cells in each of these territories. Perifascicular injection of propidium iodide around the sciatic nerve was followed by a marked cellular uptake not only in the epineurium but also in the endoneurium. The shape and position of the labeled nuclei strongly indicated that they were the nuclei of Schwann cells. Previous studies have shown that propidium iodide can be used as a retrograde tracer in neuroanatomic research.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytofluorescence localization of ethidium bromide in the nervous system of the mouse. I. Ethidium bromide: its distribution in regions within and without the blood-brain barrier after intravenous injection.

A direct fluorescence-microscopic technique was effected to determine in the central nervous system (CNS) of the mouse the distribution of ethidium bromide after intravenous (i.v.) injection. The compound was visualized in thin cryostat sections of the brain fixed by vascular perfusion through the heart with a 10% buffered formalin solution. Ethidium bromide emitted a bright red fluorescent light in model experiments. The compound could not be detected in the vessel walls or brain parenchyma of the cerebral gray and white matters after i.v. injection indicating the presence of a blood-brain barrier (BBB) phenomenon to this compound. Signs of extravasation of ethidium bromide were present in the choroid plexus, the postremal area, the Gasserian ganglion, and in the circumventricular organs of the brain (neurohypophysis, organum vasculosum lamina terminalis, and median eminence) 3 min after the i.v. injection. Intense fluorescence was present in the nucleus and the cytoplasm of the cells in these areas, located outside of the BBB. Fluorescence had disappeared 24 h after the injection. Unexpectedly, red fluorescent material was seen in the parenchyma of the olfactory lobes of some animals, indicating, possibly, the presence of ethidium bromide. Ethidium bromide is known to suppress RNA, DNA, and protein synthesis in mammalian cells and has been used previously in neuropathology for studies on myelin lesions after injury to oligodendroglial cells. It can now, by a simple fluorescence-microscopic method, be traced directly in fixed tissue. Correlations can therefore be made between localization of the compound and its cytotoxic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence-microscopic localization of in vivo injected ethidium bromide in the nervous system of the mouse.

Ethidium bromide is a compound which can suppress DNA, RNA, and protein synthesis in mammalian cells. It is a very useful tool in experimental neuropathology for studies on myelin lesions taking place in the spinal cord after injury to oligodendroglial cells following intracisternal or intraspinal administration. By using a technique described in this short original communication we can now directly trace the distribution of the compound in various cells of the central and the peripheral nervous systems after its administration to a living experimental animal. Therefore, in the future direct correlations can be made between the cellular distribution of the compound and its cytotoxic effects.

Cytotoxic effects of adriamycin on the central nervous system of the mouse--cytofluorescence and electron-microscopic observations after various modes of administration.

Adriamycin (doxorubicin) is commonly used in the treatment of malignant tumours. Adverse effects on the CNS have not been described so far, but the patients may suffer from a dose-related myocardial toxicity. Lesions have previously been observed in peripheral ganglia of experimental animals. Using a direct fluorescence microscopic method we have investigated the distribution of adriamycin in the CNS of normal mice after various modes of administration. Adriamycin, after intravenous (i.v.) injection, did not pass into the brain generally but entered the choroid plexus and circumventricular organs, namely the median eminence, postremal area, subfornical organ, organum vasculosum of the lamina terminalis, pineal gland, and neurophypophysis. After a single i.v. injection of the drug, the animals showed distinct morphological changes in three regions examined thus far, the neurohypophysis (NH), median eminence (ME), and postremal area (PA). In the NH and ME many degenerated neurosecretory axon terminals were observed. In addition, nuclear and cytoplasmic changes were seen in the pituicytes and glial cells of the ME. The PA showed severe neuronal alterations which included nucleolar segregation, rarefaction of the nuclear chromatin, and cytoplasmic changes. When the blood-brain barrier was circumvented by direct microinjection into the cerebral ventricles, the drug passed into the surrounding brain parenchyma, being detected in the nuclei of both neurons and glia. It can therefore be assumed that, when adriamycin is given to patients with a disturbance of the blood-brain barrier, the drug may spread into the brain in the same way. The blood-brain barrier can also be bypassed by injecting a substance intramuscularly or/and intradermally and letting it pass into the spinal cord or brain-stem by retrograde axonal transport. In model experiments, adriamycin was injected into the tongue and six hours later its fluorescence could be detected in the hypoglossal neurons. Animals allowed to survive for a longer period, showed selective damage to these neurons as evidenced by early nuclear changes followed by alterations in the cytoplasm.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxic effects of adriamycin on the central nervous system. Ultrastructural changes in some circumventricular organs of the mouse after intravenous administration of the drug.

Recent experimental studies have shown that the cytotoxic antibiotic adriamycin (doxorubicin) after systemic administration can enter the so-called circumventricular organs (CVO) of the brain of the mouse. The present experiments were performed to find out whether such penetration of the brain is associated with signs of neurotoxic injury. For this purpose, light- and electron-microscopic observations were carried out on three of these organs: the neurohypophysis (NH), median eminence (ME), and postremal area (PA). Pronounced widening of the extracellular space indicating the presence of edema was present in all the regions, particularly in animals examined within 3 days of injection of the drug. Many degenerated axon terminals were observed in the NH and ME. The glial cells within these regions showed rarefaction of the nuclear chromatin, nucleolar segregation, and also cytoplasmic changes. The PA presented marked cellular changes resulting in degeneration of neurons, which was most evident 30 days after the injection. Hence, regions of the CNS outside the blood-brain barrier can be reached by adriamycin after systemic administration, and the drug can induce morphological changes there. The doses of the drug used in the present experiments were comparable to those given to patients for the treatment of malignant tumors.

Cytofluorescence localization of adriamycin in the nervous system. IV. cellular uptake of the drug in peripheral nerve following various modes of injection to bypass the blood-nerve and the perifascicular barriers.

Adriamycin (Doxorubicin) is a powerful anthracyclic compound, which is widely used in the treatment of malignant diseases. In the rat a single systemic injection of the drug can induce pronounced lesions in peripheral ganglia, whereas in other parts of the peripheral nervous system (PNS) no changes have been reported. Since adriamycin can be directly traced in tissue sections by fluorescence microscopy it is very well suited for experimental studies on the relation between cytotoxic effects and distribution of the drug following various modes of administration. We have previously shown that after an intravenous (i.v.) injection there is an absence of adriamycin-induced nuclear fluorescence in the endoneurium of mouse sciatic nerve (Bigotte et al. 1982 b). This could either be due to barrier effects in endoneurial vessels and the perineurium or to a lacking capacity of the endoneurial cell population to take up and retain adriamycin. In the present study the blood-nerve and the perifascicular diffusion barriers were therefore bypassed by endoneurial microinjections of adriamycin. After this mode of administration, Schwann cells, endoneurial mast cells, endothelial cells, and pericytes became labeled. Experimental damage of these barriers induced by ligation of the nerve also resulted in a diffusion of the drug into the endoneurial area and labeling of the same cells. The absence of nuclear binding in the endoneurium of mouse sciatic nerves after i.v. injection of adriamycin is therefore most probably due to a low or absent passage of the drug from the blood into the endoneurium, i.e., a combined barrier action of endoneurial vessels and the perineurium. Other experiments with epineurial application of the drug showed that thin intramuscular (i.m.) nerve branches differ from the sciatic nerve fascicles in allowing small amounts of adriamycin to enter the endoneurium. The present observations are of interest since it can be assumed that patients receiving adriamycin as a cytostatic drug may suffer nerve lesions whenever defects of nerve barriers are present.

Cytotoxic effects of adriamycin on mouse hypoglossal neurons following retrograde axonal transport from the tongue.

We reported recently that the fluorescent, cytostatic drug, adriamycin (Doxorubicin) may reach the hypoglossal neurons by retrograde axonal transport from the nerve terminals of the tongue. The present investigation was undertaken to ascertain whether morphological changes occur in the hypoglossal neurons due to retrograde transport of adriamycin. Neuronal degeneration was observed in the hypoglossal nucleus 14 days after i.m. injection of adriamycin into the tongue. Early neuronal changes, such as rarefaction of the nuclear chromatin and segregation and fragmentation of the nucleolar components, were succeeded by cytoplasmic vacuolation, disappearance of ribosomes and other degenerative features. These observations are important from a neurotoxicologic viewpoint since they demonstrate that retrograde axonal transport may provide a route for the entry of adriamycin into the nervous system. Thus far, adriamycin appears to be the only known substance which can be traced directly in the neurons and cause their degeneration. An experimental method of damaging the motor neurons of the CNS has been introduced. A new toxic model for the investigation of experimental motor neuron disease is therefore available by the use of adriamycin.

Retrograde transport of doxorubicin (adriamycin) in peripheral nerves of mice.

Doxorubicin (adriamycin) is a fluorescent compound which is widely used in the treatment of malignant tumors due to its capacity to bind and influence the DNA in the nucleus of cells. We have now observed that the compound after injection into a skeletal muscle of adult mice is transported to the corresponding nerve cell bodies, i.e. can be used as a retrograde tracer in neuroanatomical and neuropathological research. Six hours after injection into the tongue nerve cell nuclei were labeled in the hypoglossal nuclei of the brainstem. The fluorescent tracer had the same distribution as the chromatin. Glial nuclei in the vicinity of the hypoglossal neurons were also labeled presumably due to a transfer from the neurons to the glial cells during life or during the histochemical procedure. Since doxorubicin is also neurotoxic it might be a useful tool in neurobiological research, particularly if the labeled neurons later on will show toxic effects. In this way the compound could be used in models for experimental motor neuron disease and provide a means by which retrograde fluorescent tracing and a degeneration method can be combined for studies on various neuronal systems.

Cytofluorescence localization of adriamycin in the nervous system. I. Distribution of the drug in the central nervous system of normal adult mice after intravenous injection.

By a fluorescence-microscopic technique the distribution of the antineoplastic glycoside, adriamycin (doxorubicin), was studied in the CNS of normal adult mice after i.v. injection. Doses comparable to those used in patients for the treatment of malignant diseases were used. The drug did not have access to areas of the brain within the blood-brain barrier but, except for the subcommissural organ, it was consistently localized in the nuclei of neurons and/or glial cells of the circumventricular organs (postremal area, subfornical organ, median eminence, neurohypophysis) as well as in cells of the choroid plexus and lamina cribrosa of the optic nerve. The nuclear fluorescence was accompanied by a less intense extracellular fluorescence when the survival time was shorter than 1 min after the injection. The fluorescence emitted by adriamycin was seen as early as 15 s after injection and showed its highest intensity at 1 and 15 min later. After 24 h fluorescence was no longer observed except for the ependymal zone of the median eminence. Our study thus shows that adriamycin passes from the blood into the nervous parenchyma in those areas of the brain located outside the blood-brain barrier. This finding raises the question whether in such regions there are any neurotoxicologic effects produced by the drug which have not yet been detected.

Cytofluorescence localization of adriamycin in the nervous system. II. Distribution of the drug in the somatic and autonomic peripheral nervous systems of normal adult mice after intravenous injection.

By a fluorescence-microscopic technique, the distribution of the antineoplastic glycoside adriamycin (doxorubicin) was studied in the peripheral nervous system (PNS) of normal adult mice after i.v. injection. Doses comparable to those used in patients for treatment of malignant diseases were used. The orange-red fluorescence of the drug was observed in dorsal root ganglia, in the trigeminal ganglia, and in the superior cervical sympathetic ganglia where it was preferentially accumulated in the nuclei of satellite cells. This nuclear labeling was a very quick process which occurred in the superior cervical ganglion within 15 s after the injection. Adriamycin-fluorescent nuclei were also observed in the suprarenal medulla. Fluorescent nuclei were present within the pre- and postganglionic sympathetic nerve trunks close to the superior cervical ganglion but not in the endoneurium of the trigeminal and the sciatic nerves or in the spinal nerve roots. In such structures labeled cells appeared in the connective tissue sheaths covering the nerves and the roots. No adriamycin-induced fluorescence was detected in the myenteric plexus of the intestine. Our study thus shows that i.v. injected adriamycin is distributed preferentially within areas of the PNS where the blood vessels are known to be highly permeable.