* Department of Pathology and
Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and
Division of Neurotoxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079
Received February 11, 2000; accepted May 18, 2000
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ABSTRACT |
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Key Words: ibogaine; Purkinje neuron; Bergmann astrocyte; neurodegeneration; calbindin; GFAP; NOAEL; cerebellum.
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INTRODUCTION |
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Balanced against these promising experimental results, studies by O'Hearn et al. (1993) and O'Hearn and Molliver (1993) found that ibogaine, like its structural relative, harmaline, was toxic to the cerebellum, probably via overstimulation of excitatory climbing fibers synapsing onto the Purkinje cells. Ibogaine also produced Bergmann astrocytosis and microgliosis at a dose of 100 mg/kg, intraperitoneally. Scallet et al (1996b) observed similar results at the same dose level in rats, but not in mice. Based on the neuroanatomical pattern of IBO activation of c-fos immunoreactivity, an activation of serotonin receptors in the forebrain was proposed as the initial site of ibogaine neurotoxicity (Scallet et al., 1996a). Corticofugal axons could then stimulate the inferior olive and its excitotoxic climbing-fiber pathway to the cerebellum.
The cytotoxic effects of IBO have been found predominantly in the vermis (O'Hearn et al., 1993) and simplex lobules (Molinari et al., 1996
) of the cerebellum, and were characterized by narrow longitudinal bands of degenerating Purkinje neurons and activated astrocytes (O'Hearn et al., 1993
; Scallet et al., 1996a
,b
). The toxic effects were more potent in female rats than males, as also reported by O'Callaghan et al., 1996 for the endpoint of increased GFAP content. Although O'Callaghan et al. reported increased GFAP by neurochemical analyses in forebrain areas as well as in the cerebellum, the studies cited above that employed selective histological markers of neurodegeneration have not reported damage outside the cerebellum. Since our own previous study (Scallet et al., 1996b
) carefully examined non-cerebellar areas and failed to detect neurodegeneration, the present study was restricted in focus to the cerebellum, where meaningful histological dose-response results could be expected.
Our previous studies (Scallet et al., 1996a,b
) had evaluated the neurohistological effects of ibogaine at several post-treatment intervals: 1 h, 24 h, and 7 days after dosing. The results indicated that early-immediate gene proteins such as c-fos were clearly activated, as early as at 1 h, in the cerebellum and throughout many regions of the forebrain. However, the markers of neuronal damage (silver staining and loss of calbindin immunoreactivity) and glial activation (GFAP immunoreactvity) only became responsive to treatment in the 7-day survival group. Since the aim of the present study is primarily to evaluate the dose-response characteristics of ibogaine for the production of neuronal damage, the 7-day interval was selected as optimal.
The 100-mg/kg dose that produced neurotoxic effects in several different studies by separate groups (Molinari et al, 1996; O'Hearn et al., 1993
; Scallet et al., 1996b
;) is only 23 times greater than the doses of ibogaine that have been used for rodent studies of its therapeutic properties against drug-seeking behaviors (Glick et al, 1992
). These dose levels of 3050 mg/kg are also within the range of ibogaine doses that have been used, orally, by humans in therapeutic trials (Lotsof, 1985
; Sanchez-Ramos and Mash, 1994
). Therefore, it is important to determine the neurotoxicity of ibogaine at lower dose levels. No obvious degeneration was detected by Molinari et al. (1996) following a single 40 mg/kg injection in female rats, nor was any neurotoxicity reported by Sanchez-Ramos and Mash (1994) in African green monkeys following 4 consecutive treatments with 25 mg/kg. However, to characterize the toxicity of ibogaine more completely, it is important to include a broad range of doses and to use the most sensitive histochemical methods available.
Therefore, we designed a dose-response study focusing on the female rat cerebellum, using several neurohistochemical approaches, each of which had been previously shown to be sensitive to ibogaine neurotoxicity. Our goals were to confirm the effects of 100 mg/kg ibogaine, to evaluate potential changes in the extent, distribution, occurrence, or characteristics of ibogaine lesions at lower doses, and to compare the various neurohistochemical methods for their utility at detecting the neurotoxicity of low doses of ibogaine.
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MATERIALS AND METHODS |
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Drug and dose exposure.
Ibogaine hydrochloride (HCl), obtained from Sigma Chemical Co. (St. Louis, MO), was dissolved in deionized water as previously described (Scallet et al., 1996b). The dosing solution resolved as a single peak by HPLC and was stable for at least 24 h. No confirmation of the structure was performed. Group 1 animals were injected intraperitoneally (ip) with 10 ml/kg of saline. Group 2, 3, 4, and 5 animals were injected with ibogaine solutions of 2.5 mg/ml, 5 mg/ml, 7.5 mg/ml, or 10 mg/ml respectively. Each rat was injected with a volume of 10-ml/kg body weight, resulting in doses of 25, 50, 75, or 100 mg/kg of ibogaine. All animals were injected within 2 h of the preparation of the dosing solutions.
Behavioral observations.
Although a formal Functional Observational Battery was not performed, the animals were observed periodically after treatment for their general activity and demeanor. For the first 8 h after dosing, each rat was observed at a frequency of once per h. A second observation was conducted approximately 24 h after dosing. The observer was not blind as to the identity of the treatment groups.
Perfusion and fixation.
The animals were allowed to survive until 7 days after ibogaine (or saline) treatment, a time interval suitable for studying Purkinje cell neurodegeneration, as determined previously (Scallet et al., 1996b). The general health and disposition of the animals was good during this period of time as indicated by their body-weight data. All groups had gained comparable amounts of weight by 7 days after dosing (controls, 14 g; 25 mg/kg IBO, 4 g; 50 mg/kg IBO, 19 g; 75 mg/kg IBO, 14 g; 100 m/kg IBO, 10 g) Then, a transcardial perfusion was performed as described by Scallet, 1995. Briefly, sacrifice was by sodium pentabarbital anesthesia (100 mg/kg, ip), followed by intracardiac perfusion using a peristaltic pump set to a flow rate of 35 ml/min. Following a flush of 45 ml saline, about 500 ml of 4% formaldehyde in 0.1 M phosphate buffer was perfused through the vasculature. After overnight post-fixation in the perfusate, cerebella of the animals were cut with a VibratomeTM (Technical Products, Inc., St. Louis, Mo) into 50-micron coronal sections.
Histochemistry: Nadler-Evenson degeneration-selective silver stain method (N-E).
The above procedure is utilized for selectively staining degenerating neurons and is described in detail by Scallet, 1995 as modified from Nadler and Evenson, 1983. Briefly, sections were pretreated with a dilute alkaline solution of ammonium nitrate (pretreatment), followed by 0.50% silver nitrate in alkaline ammonium nitrate (impregnation). After impregnation, sections were rinsed with a sodium carbonate/ammonium nitrate solution (washing), and finally developed in citric acid in dilute formaldehyde/ethanol (development). The sections were mounted on gelatin-subbed glass slides following the development. After drying, they were fixed with 0.5% acetic acid, dehydrated through an ascending series of ethanol concentrations (75%, 95%, and 100%) to xylene, and cover-slipped with permount.
Histochemistry: Anti-calbindin immunocytochemical stain.
Calcium binding proteins have been used as marker proteins for neurons (Anderssen et al., 1993; Baimbridge et al., 1992
). The immunostaining procedure used to localize calbindin followed the description of Scallet (1995), as modified from the indirect antibody peroxidase-antiperoxidase method of Sternberger (1982). Briefly, sections were incubated overnight at 4°C in a 1:500 dilution of a mouse monoclonal primary antibody to calbindin (Sigma). Then they were placed in a 1:200 dilution of goat anti-mouse secondary antibody (Jackson Immunoresearch Lab, West Grove, PA) for 1 h. After the secondary antibody incubation, sections were washed and placed in a 1:200 dilution of mouse peroxidase-antiperoxidase solution (Jackson) for another h. Finally, they were placed for 10 min in a DAB (3,3`-diaminobenzidine tetrahydrochloride, Sigma) and hydrogen peroxide solution.
Histochemistry: Anti-glial fibrillary acidic protein (GFAP) immunocytochemical stain.
We conducted GFAP immunohistochemistry exactly as with the anti-calbindin staining above, except that we utilized a polyclonal rabbit antisera to GFAP (Sigma) as the primary antibody. The secondary antibody was goat anti-rabbit IgG (1:100) (Sigma), and rabbit PAP (1:50) (Sigma) was used.
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RESULTS |
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Nadler-Evenson Degeneration-selective Silver Stain
The above method specifically stains degenerating neuronal elements, such as dendrites and axons, as dark, black varicose fibers against a pale yellow background. Degenerating cell bodies are seen as slightly shrunken black profiles (Nadler and Evenson, 1983; Scallet, 1995
). No detectable morphological changes were observed in sections from 25 mg/kg animals (Figs. 1A and 1B
), which were indistinguishable from control sections. With 50 mg IBO/kg, 2 of the 6 animals showed small regions of argyrophilic degenerating axons coursing through the white matter of the cerebellum and surrounding the deep cerebellar nuclei representing axon and terminal degeneration (Fig. 1C
), but few degenerating neuronal cell bodies were observed in the cerebellar cortex (Fig. 1D
). All 6 of the animals that received 75 mg IBO/kg exhibited degenerating axons surrounding the deep cerebellar nuclei (Fig. 1E
). Multiple, but relatively narrow, bands of degenerating Purkinje cell bodies were also observed in each 75-mg/kg rat (Fig. 1F
). All 100-mg IBO/kg rats showed thick bundles of intensely stained degenerating axons throughout the regions surrounding the deep cerebellar nuclei. In 100-mg IBO/kg animals, wide bands (Fig. 1H
) of degenerating Purkinje cell bodies were stained black, with their heavily stained degenerating dendrites extended outward into the molecular layer.
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DISCUSSION |
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The Nadler-Evenson degeneration-selective silver stain method revealed a dose-response relationship in ibogaine-induced Purkinje neuronal degeneration. Effects on the numbers of degenerating axons and neurons per animal, and the fraction of animals per group that had sustained damage were each related to dose (Fig. 1, Table 1
). The degeneration first appeared as distinct axonal tracts terminating in the deep cerebellar nuclei of animals receiving as little as 50 mg IBO /kg. This dose is very close to that used for interrupting morphine self-administration in animal models (Glick et al. 1991
). It is important to recognize that ibogaine should be considered neurotoxic at the 50-mg/kg dose, despite our failure to observe many degenerating Purkinje cell bodies. Since the principal output of the Purkinje neurons is to the deep cerebellar nuclei, even a few degenerating cell bodies may be detected here by their degenerating axons. Since degenerating axons become concentrated here, it also follows that the deep cerebellar nuclei should be considered as an important and sensitive site for neurotoxicological evaluations where Purkinje cell toxicity is suspected.
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A dose-related response was also observed with the anti-GFAP method (see Fig. 3, Table 1
). Bands of intense GFAP immunostaining representing an increased degree of reactive astrocytosis by Bergmann glia were detectable even at doses as low as 50 mg/kg, whereas the Nadler-Evenson silver staining approach detected only degenerating Purkinje cell axons and their terminals, but few or no neuronal perikarya. In the present study, GFAP staining in the 50-mg IBO/kg animals showed bands that were relatively wider and more frequently occurring than the occasional pale band, the width of one Purkinje neuron, which were observed in anti-calbindin-stained sections from the same animal (Fig. 3B
). These observations of the 50-mg/kg animals reinforce the idea that GFAP staining is particularly sensitive for detecting ibogaine neurotoxicity in the cerebellum.
Our present study further supports the several previous observations of ibogaine neurotoxicity at doses of 100 mg/kg ip (Molinari et al., 1996; O'Hearn and Molliver, 1993
, 1997
; O'Hearn et al, 1993
O'Hearn et al, 1995, Scallet et al. 1996a
,b
). However, our present investigations have also demonstrated the dose-response relationship, for each of 3 different neuropathological techniques by which ibogaine produces various signs of Purkinje cell damage. A dose of 25 mg IBO/kg was the highest level at which no-observable-adverse-effects (NOAEL) of ibogaine occurred in rats observed with any of the techniques.
The most sensitive procedures seemed to be immunohistochemistry for GFAP in the cerebellar cortex and the N-E silver stain for degenerating axons in the deep cerebellar nuclei. The N-E method has been widely accepted to provide evidence for neuronal death or damage, whereas Alzheimer's Type II gliosis may also occur under other circumstances, such as hyperammonemia (Scallet, 1995). Since we have not yet examined the time course of the responses of the various biomarkers between 1 and 7 days, it should be considered that their apparent relative sensitivity to ibogaine's cerebellar toxicity might be different at other intervals. However, clear evidence of Purkinje neuronal degeneration appeared with the N-E silver method at doses as low as 50 mg IBO/kg in our study.
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NOTES |
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REFERENCES |
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Aschner, M. (1998). Astrocytic functions and physiological reactions to injury: The potential to induce and /or exacerbate neuronal dysfunction (a forum position paper). Neurotoxicology 19, 717.[ISI][Medline]
Baimbridge, K. G., Celio, M. R., and Rogers, J. H. (1992). Calcium-binding protein in the nervous system. Trends Neuroscience 15, 303308.[ISI][Medline]
Dzoljic, E. D., Kaplan, C. D., and Dzoljic, M. R. (1988). The effect of ibogaine on naloxone-precipitated withdrawal in chronic morphine-dependent rats. Arch. Intl. Pharmacodyn. Ther. 294, 6470.
Eng, L. F., and Ghirnikar, R. S. (1994). GFAP and astrogliosis. Brain Pathol. 4, 229237[ISI][Medline]
Glick, S. D., Rossman, K., Rao, N. C., Maisinneuve, I. M., and Carlson, J. N. (1992). Effect of ibogaine on acute signs of morphine withdrawal in rats: Independence from tremor. Neuropharmacology. 31, 497500.[ISI][Medline]
Glick, S. D., Rossman, K., Steindorf, S., Maisonneuve, I. M., and Carlson, J. N. (1991). Effects and after-effects of ibogaine on morphine self-administration in rats. Eur. J. Pharmacol. 195, 341345.[ISI][Medline]
Graeber, M. B., Streit, W. J., and Kiefer, R. (1986). Astrocytes increase in GFAP during retrograde changes of facial motor neurons. J. Neuroimmunology 27, 121132.[ISI][Medline]
Kaplan, C. D., Ketzer, E., De Jong, and De Veries, M. (1993). Researching a state of wellness: Multistage exploration in social neuroscience. Soc. Neurosci. Bull. 6, 69.
Lotsof, H. S. (1985). Rapid method for interrupting the narcotic addiction syndrome. U.S. Patent No. 4,499,096.
Lotsof, H. S. (1991). Rapid method for interrupting or attenuating nicotine/tobacco dependency syndromes. U. S. Patent No. 5,125,994.
Lotsof, H. S. (1995). Ibogaine in the treatment of chemical dependency disorders: Clinical perspectives. Bull. MAPS. 5, 1627.
Maisonneuve, I. M. (1992). Ibogaine and brain dopamine systems: Interaction with drugs of abuse. Dissertation Abstracts International 52, 5773-B.
Molinari, H. H, Maisonneuve, I. M., and Glick, S. D. (1996). Ibogaine neurotoxicity: A re-evaluation. Brain Res. 737, 255262.[ISI][Medline]
Nadler, J. V., and Evenson, D. A. (1983). Use of excitatory amino acid to make axon-sparing lesions of hypothalamus. Methods Enzymol. 103, 393400.[ISI][Medline]
O'Callaghan, J. P. (1991). Assessment of neurotoxicity: Use of glial fibrillary acidic proteins as a biomarker. In Biomedical and Environmental Sciences (L. W. Chang, Ed.). Academic Press, San Diego.
O'Callaghan, J. P., Rogers, T. S., Rodman, L. E., and Page, J. G. (1996). Acute and chronic administration of ibogaine to the rat results in astrogliosis that is not confined to the cerebellar vermis. Ann. N Y Acad. Sci. 801, 205217.[Abstract]
O'Hearn, E., Long, D. B., and Molliver, I. M. (1993). Ibogaine induces glial activation in parasagittal zones of the cerebellum, NeuroReport,, 4, 299302.[ISI][Medline]
O'Hearn, E., and Molliver, M. E. (1993). Degeneration of Purkinje cells in parasagittal zones of the cerebellar vermis after treatment of ibogaine and harmaline. Neuroscience 55, 303310.[ISI][Medline]
O'Hearn, E., and Molliver, M. E. (1997). The olivocerebellar projection mediates ibogaine-induced degeneration of Purkinje cells: A model of indirect, trans-synaptic excitotoxicity. J. Neurosci. 17, 88288841.
Sanchez-Ramos, J., and Mash, D. (1994). Ibogaine research update: Phase I human study. Bull MAPS 4, 11.
Scallet, A. C. (1995). Quantitative morphometry for neurotoxicity assessment. In Neurotoxicology: Approaches and Methods (Chang, W. L. and Slikker, W., Eds.), pp. 99121. Academic Press, San Diego.
Scallet, A. C., Ye, X., and Ali, S. F. (1996a). NOS and fos in rat and mouse brain regions: Possible relation to ibogaine-induced Purkinje cell loss. Ann. N Y Acad. Sci. 801, 227238.[Medline]
Scallet, A. C., Ye, X., Rountree, R., Nony, P., and Ali, S. F. (1996b). Ibogaine produces neurodegeneration in rat, but not mouse, cerebellum. Ann. N Y Acad. Sci. 801, 217226.[Medline]
Sternberger, L. A. (1982). Immunocytochemistry, 2nd ed., pp. 187. Wiley Press, New York.