Z. Zellforsch. 141, 517--527 (1973) © by Springer-Verlag 1973

Fluorescence Histochemical and Uhrastructural Observations on the Effects of Intravenous Injections

of Vinblastine on Noradrenergic Nerves*

Terence Benne t t

Department of Physiology, Nottingham University, Medical School, Nottingham, England

James L.S. Cobb

Garry marine Laboratory, St. Andrews, Scotland

Torbj6rn Malmfors

Department of Histology, Karolinska Instituter, Stockholm, Sweden

Received April 16, 1973

Summary. Fluorescence histochemical and ultrastructural observations were made on noradrenergic nerves in chicks, following intravenous injections of vinblastine. The drug caused a reversible blockade of the axoplasmic transport of catecholamines in non-terminal axons. Some terminal fibres showed a loss of fluorescence 48 hours after injections of animals with 10 mg/kg vinblastine, and ultrastructurally there were signs of neural necrosis.

Key words: Noradrenergic nerves - - Vinblastine - - Axoplasmic transport - - Degeneration - - Fluorescence and electron microscopy.

Introduction

Recently, there have been a n u m b e r of studies concerned with the effects of the mitot ic inhibitors, colchicine and vinblast ine, on noradrenergic neurones (eg. DahlstrSm, 1968, 1970, 1971; Keen and Livingston, 1970, 1971; Hokfe]t and DahlstrSm, 1971; Banks et al., 1971). The major i ty of these studies dealt with the changes occurring in cell bodies and non- te rmina l axons when the drugs were applied directly to those regions. However, Keen and Livingston (1970, 1971) found tha t in t ravenous inject ion of v inblas t ine into rats caused a fall in the noradrenal ine conten t of the hear t and vas deferens. Since it has long been known tha t eolchicine may cause degenerat ion of axons (eg. Angevine, 1957), the present s tudy was carried out to invest igate the morphological effects of v inblas t ine on noradrenergie nerves, when injected int ravenously. A pre l iminary account of these results has been given (Bennet t et al., 1971 a).

Materials and Methods White leghorn chicks, 1 to 2 weeks old were used in this study. Vinblastine sulphate

(Velbe, Lilly) was dissolved in sterile saline and injected into a wing vein. Preliminary ob- servations showed that doses of vinblastine less than 10 mg/kg had very variable effects on noradrenergic nerves; for this reason all observations were made after injection of 10 mg/kg

* Some of the work described in this paper was carried out in the Department of Zoology, Melbourne University. We are grateful to professor G. Burnstock for use of facilities, and the National Heart Foundation of Australia for financial assistance.

518 T. Bennett et al.

of the drug. In each experimental group there were 9 animals; 6 were injected with vinblastine and 3 were injected with saline. Groups of animals were killed, 1, 8, 15, 24 and 48 hours after injection. Tissues (oblique septum; vena cava; coccygeomesenteric vein; posterior mesenteric artery; left atrium) from 2 treated animals were prepared immediately for the fluorescence histochemical localisation of biogcnic amines (see Bennett and Malmfors, 1970). The tissues from another 2 animals injected with vinblastine were excised and incubated in a physiological saline solution at 37°C for 30 minutes, after the large nerve trunks had been compressed with fine forceps. This procedure permits the detection of axoplasmic transport of catecholamines (see Bennett and Malmfors, 1970). The remaining 2 treated animals had their tissues removed and processed for electron microscopic examination (Cobb and Beunet, 1971). Tissues from the 3 saline-injected control animals were processed together with those of the 3 groups of vin- blastine-treated animals described above.

Results

Fluorescence Histochemistry

In the tissues from control animals, brightly fluorescent catecholamine accumulations were seen above the points of compression of the large nerve bundles. Such accumulat ion was not seen in tissues taken from animals t reated one or eight hours previously with 10 mg/kg vinblastine. Examinat ion of tissues 15 hours after vinblastine injection showed some indications of axoplasmic t ranspor t of catecholamines. But between 15 and 48 hours it was difficult to judge the increase of fluorescence above compressions in nerve trunks, since the nerve t runks when not compressed showed a progressive increase in fluorescence intensi ty during this time. I n general, however, it appeared tha t injection of vinblastine reversibly blocked axoplasmic t ranspor t of cateeholamines for at least 8 hours. During the time tha t axoplasmic t ranspor t of catecholamines was blocked (ic. up to 8 hours) there were no obvious changes in the fluorescence morphology of the noradrenergic nerves. There were slight changes in the ap- pearances of nerves in tissue taken 15 and 24 hours after injection of vinblastine, but the changes were most marked 48 hours after t reatment . The effects of the drug varied somewhat f rom tissue to tissue, and so are considered separately here:

Oblique Septum. The plexus of termina] and non-terminal noradrenergic axons associated with the oblique septum (Bennett and Malmfors, 1970) was readily detected in control tissues (Fig. 1). However, 48 hours after injection of vinblastine only few, faintly fluorescent termina] fibres were seen (Fig. 2).

Posterior Meseuteric Artery. Under normal conditions, the non-terminal axon bundles accompanying the posterior mescnteric ar tery were obscured by the brightly fluorescent perivascular plexus (Fig. 3, see Bennet t et al., 1973). For ty- eight hours after t rea tment with vinblastine there was a dramat ic loss of fluores- cence from the perivascular plexus (Fig. 4), while the non-terminal axon bundles showed signs of catecholamine accumulat ion over much of their lengths. This picture is similar to tha t seen after t rea tment with 6-hydroxydopamine (Fig. 5, see Bennet t et al., 1973).

Coceygeomesenterie Vein. I n unt rea ted animals, the terminal fibres and non- terminal axon bundles, associated with the coccygeomesenteric vein, formed a conspicuous plexus (Fig. 6). After t rea tment with vinblastine, the plexus of ter-

Vinblastine and Noradrenergic Nerves 519

Fig. 1. Oblique septum from an animal injected with saline. Note the loose-meshed plexus of non-terminal axon bundles (NT), and the occasional, varicose terminal fibres. Running across the picture is the perivaseular plexus of an arteriole (ART). Stretch preparation. Calibration

100

Fig. 2. Oblique septum from an animal injected 48 hours previously with vinblastine. A few, scattered terminal fibres (T) are seen, bu t are only faint ly fluorescent. The non-specific fluores- cence of the background is more obvious t han in the control tissues, and the blood vessels (ART) appears darker, due to the presence of erythrocytes within them. Note t h a t the peri-

vascular plexuses are not detectable. Stretch preparation. Calibration 100 t~

520 T. Bennett et al.

Figs. 3--5

Vinblastine and Noradrenergic Nerves 521

minal f ibres was ve ry pa tchy , while the non- te rmina l axon bundles showed a m a r k e d increase in fluorescence in t ens i ty over much of the i r lengths (Fig. 7).

Heart. The inne rva t ion of the left a t r ium, in control animals , consisted of a loose- meshed plexus of varicose and non-var icose nerve fibres (Fig. 8). Af te r t r ea t - ment , 48 hours p rev ious ly wi th vinblas t ine , the ground plexus was more i r regular t han normal , bu t the non- t e rmina l axon bundles d id not show a m a r k e d increase in f luorescence in t ens i ty (Fig. 9).

Electron Microscopy

As with the fluorescence his tochemical findings, the most s t r ik ing ul t ra- s t ruc tu ra l changes were seen 48 hours af ter t r e a t m e n t wi th vinblas t ine . A t this t ime enormous numbers of lysosome-l ike bodies were seen in some axons and Schwann cells (Fig. 10). Pa r t i cu l a r l y in non- te rmina l axons, there were present large numbers of m e m b r a n e - b o u n d e d granu la r bodies (Fig. 11). I n m a n y axon profiles, necrot ic mi tochondr ia were seen, bu t i t was not clear whether these were re la ted to the m e m b r a n e - b o u n d e d granu la r bodies. Whi le some axon profiles showed signs of necrosis, o thers appea red normal (Figs. 10, 11), and in some, neuro tubules were presen t (Fig. 10). No extens ive s t u d y was made of the vesicle popula t ions in axons following t r e a t m e n t wi th vinblast ine . However , subject ively , there appea red to be ve ry few dense-cored vesicles present in the axons following t r e a t m e n t wi th vinblast ine .

Discussion

Vinblas t ine appl ied d i rec t ly to non- te rmina l noradrenergic axons appears to p reven t the in t r aneurona l t r a n s p o r t of ca techolamines (DahlstrSm, 1970, 1971; H6kfe l t and Dahls t rSm, 1971). However , p rox ima l to the site of app l ica t ion of the drug there is a m a r k e d accumula t ion of ca techolamines ; thus, the s i tua t ion is comparab le to t h a t seen when nerve t runks are l iga ted mechanica l ly (H6kfel t and Dahls t r6m, 1971). This implies t h a t topical t r e a t m e n t wi th v inblas t ine does not interfere wi th ca techolamine t r anspo r t general ly, bu t only at the site where i t is applied.

Fig. 3. Posterior mesenteric artery from an animal injected with saline. The bright fluorescent terminal fibres are aligned with the longitudinal axis of the vessel, and tend to obscure the loose perivascular plexus of non-terminM axon bundles. Some faintly-fluorescent non- terminal axon bundles (NT) are seen accompanying the vessel. Stretch preparation. Cali-

bration 100

Fig. 4. Posterior mesenteric artery from an animal injected with vinblastine 48 hours pre- viously. Note that the majority of terminal fibres are no longer detectable, although, in locali- sed regions, some remain. The non-terminM axon bundles (NT) are now readily seen, due to the loss of terminal fibres and to the marked increase in fluorescence intensity of the axon bundles.

Stretch preparation. Calibration 100

Fig. 5. Posterior mesenteric artery from an animal injected 24 hours previously with 6-hy- droxydopamine (100 mg/kg). Note the loss of terminal fibres and the increase in fluorescence intensity of the non-terminal axon bundles; compare with Fig. 4. Stretch preparation. Cali-

bration 100

522 T. Bennet t st al.

Fig. 6. Coccygeomesenteric vein from an animal injected with saline. There is a close-meshed plexus of varicose terminal fibres; non-terminal axon bundles (arrow) are readily seen, and, in some places, appear as br ight as the terminals (upper right) Stretch preparation. Calibration

100 Fig. 7. Coccygeomesenteric vein from an animal injected 48 hours previously with vinblastine. Note the massive increase in fluorescence intensi ty of the large nerve t runks and the non- terminal axon bundles (arrows). In the background the plexus of terminal fibres appears very patchy, al though some remaining fibres have a normal appearance. Stretch preparation.

Calibration 100

Vinblastine and Noradrenergic N*erves 523

Fig. 8. Left a t r ium from an animal injected with saline. A loose-meshed plexus of terminal fibres and non-terminal axons is associated with the atrial musculature. Stretch preparation.

Calibration 100 ~x

Fig. 9. Left a t r ium from an animal injected 48 hours previously with vinblastine. The ter- minal plexus appears somewhat patchy, and the large nerve t runks show some signs of cate- eholamine accumulation, bu t the picture is not greatly different from t h a t seen in control

animals (see Fig. 8). Stretch preparation. Calibration 100

34 z. Zellforseh., Bd. 141

524 T. Bennett et al.

Fig. 10. Electron micrograph showing axon profiles associated with the iifferior vena cava from an animal injected 48 hours previously with vinblastine. A large axon profile containing numerous lysosome-like bodies (arrow) is seen; the Schwann cell associated with this axon contains numerous dense inclusions. Axon profiles elsewhere appear normal, and, in some,

neurotubules are distinguishable. Calibration 1 ix

Fig. 11, Electron micrograph showing axon bundle in the outer coat of the inferior vena eava from an animal injected 48 hours previously with vinblastine. Large, electron-dense bodies are seen in some axons (arrows) and Schwann cells (double arrow). In some cases the sub- structure of these membrane-bounded bodies suggests they might be necrotic mitoehondria.

Calibration 0.5 ix

Vinblastine and Noradrenergic Nerves 525

In the present study, intravenous injections of vinblastine were found to cause a reversible blockade of catecholamine transport in nerve trunks. Inter- estingly, however, the ult imate effect of such t reatment was comparable to that seen following local application, ie. marked eateeholamine accumulation in cell bodies and non-terminal axons. These similarities might be accounted for in a variety of ways, but the most likely explanation is that, during blockade of axoplasmie transport, irreversible changes occurred in the terminal noradrenergic axons, and, when axoplasmic transport was re-established, catecholamines accumulated above the site of lesion of the terminal fibres. I t is possible tha t vinblastine has a direct toxic action on noradrenergie nerve terminals, but it is more likely tha t the metabolic and nutritive demands of the terminal fibres make them susceptible to a transient blockade of axoplasmic transport. Such a suggestion requires that vinblastine should prevent some essential factor from reaching the terminals.

In mammals there is evidence tha t the drug interferes with the transport of organelles other than noradrenaline storage vesicles (ttSkfe]t and DahlstrSm, 1971), and may cause precipitation of various proteins (Wilson et al., 1970).

The fluorescence histochemical observations made in the present work show that the changes seen after vinblastine are very similar to those seen after treat- ment with 6-hydroxydopamine. The lat ter is a drug that causes degeneration of terminal noradrenergie nerves in chicks (Bennett et al., 1970, 1973), but, since it does not block axoplasmic transport (Bennett et al., 1971 b), there is a concurrent accumulation of eatecholamines in the surviving non-terminal axons. Degener- ation of terminal fibres following t reatment with 6-hydroxydopamine is signalled by a loss of eatecholamine fluorescence and marked ultrastruetural modifications (Bennett et al., 1970); similar, although less marked, changes were seen in the present study. These findings together indicate that vinblastine may cause degeneration of some terminal axons. The problem of the site of action and ulti- mate effect of vinblastine t reatment on noradrenergic nerves could be further investigated by examination of the changes in terminal fibres following local applications of the drug to the parent cell bodies.

I t is clear from the present s tudy that intravenously administered vinblastine has differential effects on noradrenergic nerves in different tissues, and even on nerves within the same tissues. Similar effects are seen following t reatment of chicks with 6-hydroxydopamine (Bennett et al., 1973), but in that instance it is not clear how far differential uptake of the drug by the different nerves contributes to the variable effects (Bennett et al., 1973). There is no evidence to suggest tha t vinblastine is preferentially accumulated by noradrenergie nerves, thus the variability in effects seen in the present s tudy must be at tr ibuted to other factors. I t may be that these regional variations reflect differences in the concentrations of the drug reaching the different nerves, due to variations in vaseularity and/or blood/nerve barriers. Another possibility is that the terminal fibres that apparently degenerated after t reatement were the most active and hence, metabolically, most demanding; these then would be more susceptile to a transient blockade of axoplasmic transport. Certainly there are indications tha t noradrenergic neurones vary widely with regard to their metabolic states (tISkfelt and Dahl- strSm, 1971).

34*

526 T. Bennett et al.

A more extended investigation is now required of the fluorescence histo- chemistry, ultrastructure and physiology of noradrenergic nerves following treat- ment with vinblastine, particularly in view of the extensive use of this drug in cancer chemotherapy.

Addendum

In a recent paper [DahlstrSm, A., tt/~ggendal, J., Linder, A.: Degeneration contraction after local vinblastine treatment of superior cervical ganglia. Europ. J. Pharmaeol., 21, 41-45 (1973)], findings were reported which corroborate the present observations. DahlstrSm etal., (1973) did not, however, distinguish differential effects of vinblastine on terminal fibres and non-terminal axons.

It is of particular relevance to the present discussion that the metabolism of vinblastine has recently been implicated in its action on noradrenergic nerves [Cheney, D.L., ttanin, I., Massarelli, R., Trabuechi, M., Costa, E.: Vinblastine and vincristine: a study of their action on tissue concentrations of epinephrine, norepinephrine and acetylcholine. Ncuropharmaeol. 12, 233-238 (1973)]. The findings of Cheney et al. (1973) suggest that noradrenergic neurones which metab- olise vinblastine rapidly would be most susceptible to the destructive effects of this drug. However, DahlstrSm et al. (1973) found that terminal fibres, innervating the iris, degenerated after local application of vinblastine to the superior cervical ganglion. In this situation it is unlikely that the drug, or a mctabolite, could be exerting its effects directly on the terminal fibres. It is probable, as suggested above, that interference with axoplasmic transport contributes greatly to the effect of vinblastine on noradrenergie nerve terminals.

References

Angevine, J. D.: Nerve destruction by colchicine in mice and golden hamsters. J. exp. Zool. 136, 363-391 (1957).

Banks, P., Mayor, D., Mitchell, 3I, Tomlinson, D. R.: Studies on the translocation of nor- adrenaline-containing vesicles in post-ganglionic sympathetic neurones in vitro, inhibition of movement by colchicine and vinblastine and evidence for the involvement of axonal microtubu]es. J. Physiol. (Lond.) 216, 625-639 (1971).

Bennett, T., Burnstock, G., Cobb, J.L.S., Malmfors, T.: An ultrastructural and histochemical study of the short-term effects of 6-hydroxydopamine on adrenergic nerves in the domestic fowl. Brit. J. Pharmaeol. 38, 802-809 (1970).

Bennett, T., Cobb, J. L. S., Malmfors, T.: The effects of intravenous injections of vinblastine on adrenergic nerves. J. Physiol. (Loud.) 217, 26-27P (1971a).

Bennett, T., Cobb, J.L.S., Mahafors, T.: Differential effects of 6-hydroxydopamine on terminal and non-terminal axons of adrenergic neurones. Brit. J. Pharmacol. 43, 445P446P (1971b).

Bennett, T., Malmfors, T.: The adrenergic nervous system of tile domestic fowl (Gallu8 domesticus (L.)). Z. Zellforsch. 106, 22-50 (1970).

Bennett, T., Malmfors, T., Cobb, J.L.S.: A fluorescence histochemical investigation of the degeneration and regeneration of noradrenergic nerves in the chick following treatment with 6-hydroxydopamine. Z. Zellforsch. in press (1973).

Cobb, J.L.S., Bennett, T.: An electron microscopic examination of the short-term effects of 6-hydroxydopamine on the peripheral adrenergic nervous system. In: 6-hydroxydop- amine and catecholamine neurons (Malmfors, T., Thoenen, It., eds.), p. 33-46. Amsterdam: North-Holland Pub]. Co. 1971.

DahlstrSm, A.: Effect of colchicine on transport of amine storage granules in sympathetic nerves of the rat. Europ. J. Pharmacol. 5, 111-112 (1968).

Vinblastine and Noradrenergie Nerves 527

DahlstrSm, A. : The effects of drugs on axonal transport of amine storage granules. In: New aspects of storage and release mechanisms of eateeholamines (Schtimann, H. J., Kroneberg, G., eds.), Bayer Symposium II, p. 20-36. Berlin-Heidelberg-New York: Springer 1970.

Dahlstr6m, A.: Axoplasmic transport (with particular respect to adrenergie neurons). Phil. Trans. B 261, 325-358 (1971).

HSkfelt, T., DahlstrSm, A.: Effects of two mitosis inhibitors (eolehicine and vinblastine) on the distribution and axonM transport of noradrenaline storage particles, studied by fluores- cence and electron microscopy. Z. Zellforseh. 119, 460482 (1971).

Keen, P., Livingston, A.: Decline of tissue noradrenaline under the influence of a mitotic inhibitor. Nature (Lond.) 227, 967-968 (1970).

Keen, P., Livingstone, A.: Intraneuronal transport of noradrenaline in the rat. In: Subcellular organization and function in endocrine tissues (Heller, H., Lederis, K., eds.). Mem. Soc. Endoer. 19, 671-679 (1971).

Wilson, L., Bryan, J., Ruby, A., Mazia, D.: Precipitation of proteins by vinblastine and calcium ions. Proc. nat. Acad. Sci. (Wash.) 66, 807-814 (1970).

Dr. T. Bennett Department of Physiology Nottingham University Medical School Nottingham NG 7 2 RD England