Toxicological Sciences 57, 95-101 (2000)
Copyright © 2000 by the Society of Toxicology
Neurotoxicology |
A Dose-Response Study of Ibogaine-Induced Neuropathology in the Rat Cerebellum
,
,,,1
* 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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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ibogaine (IBO) is an indole alkaloid from the West African shrub, Tabernanthe iboga. It is structurally related to harmaline, and both these compounds are rigid analogs of melatonin. IBO has both psychoactive and stimulant properties. In single-blind trials with humans, it ameliorated withdrawal symptoms and interrupted the addiction process. However, IBO also produced neurodegeneration of Purkinje cells and gliosis of Bergmann astrocytes in the cerebella of rats given even a single dose (100 mg/kg, ip). Here, we treated rats (n = 6 per group) with either a single ip injection of saline or with 25 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg of IBO. As biomarkers of cerebellar neurotoxicity, we specifically labeled degenerating neurons and axons with silver, astrocytes with antisera to glial fibrillary acidic protein (GFAP), and Purkinje neurons with antisera to calbindin. All rats of the 100-mg/kg group showed the same pattern of cerebellar damage previously described: multiple bands of degenerating Purkinje neurons. All rats of the 75-mg/ kg group had neurodegeneration similar to the 100-mg/kg group, but the bands appeared to be narrower. Only 2 of 6 rats that received 50 mg/kg were affected; despite few degenerating neuronal perikarya, cerebella from these rats did contain patches of astrocytosis similar to those observed with 75 or 100 mg/kg IBO. These observations affirm the usefulness of GFAP immunohistochemistry as a sensitive biomarker of neurotoxicity. None of the sections from the 25-mg/kg rats, however stained, were distinguishable from saline controls, indicating that this dose level may be considered as a no-observable-adverse-effect level (NOAEL).
Key Words: ibogaine; Purkinje neuron; Bergmann astrocyte; neurodegeneration; calbindin; GFAP; NOAEL; cerebellum.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ibogaine is a psychoactive indole alkaloid found in the West African shrub, Tabernanthe iboga. It has been proposed as a possible therapeutic aid for interrupting addictive cravings of various sorts (Glick, et al, 1992
). Both anecdotal reports in humans and studies of animal models have claimed anti-addictive properties against cocaine, morphine, amphetamine, alcohol, and nicotine (Dzoljic et al, 1988; Kaplan, et al., 1993; Lotsof, 1985, 1991, 1995; Maisonneuve, 1992).
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 2–3 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 30–50 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and groups.
Thirty female adult Sprague-Dawley rats (6 months-of-age, weighing 319.0 ± 3.8 grams, mean ± SEM, just prior to dosing) obtained from the NCTR breeding colony, were individually housed under controlled environmental conditions (temperature 22°C, relative humidity 50%, 12-h light:dark cycle with lights out at 1800 h) in plexiglass cages with wood-chip bedding. Animal care was in an AAALAC-approved vivarium, according to the Guide for the Care and Use of Laboratory Animals published by the National Research Council, NIH, Publication #85–23. Rat chow (Ralston-Purina, St. Louis, MO) was available ad libitum. The animals were randomly divided into 5 groups (n = 6 per group).
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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects on Motor Behavior by Ibogaine Treatment
After about 3 min following ip injection of ibogaine, rats displayed a series of motor behavioral changes that included tremor, ataxia, movement abnormalities, and hypotonia. No behavioral abnormalities were observed in the control animals. In 25-mg IBO/kg rats, a high-frequency tremor of the trunk, head, and limbs was detected in 4 of 6 animals. This tremor was only a transient phenomenon and the animals appeared to be normal again after about 20–40 min. With 50 mg IBO/kg, a similar tremor was observed in all rats, followed by abnormal movements, including extensions of the head, repetitive facial movements, and rapid "patting" of the forelimbs. Three of the 6 50-mg/kg animals recovered from these effects after about 3 h. The rest of the 50-mg/kg group appeared normal when observed after 24 h. Following 75 mg IBO/kg, the motor behavioral abnormalities were more severe. All 6 animals showed ataxia and a period of marked hypotonia as described by O'Hearn and Molliver (1997), but recovered by 24 h. With 100 mg IBO/kg, similar but more intense behavioral effects occurred, but this group also appeared normal when observed 24 h after dosing.
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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Anti-calbindin Immunocytochemistry
Calbindin is a calcium-binding protein that is primarily associated with Purkinje neurons in the cerebellum (Anderssen et al., 1993
). The cytoplasm of normal Purkinje cell bodies and their dendrites were strongly reactive with calbindin antibodies, while other cerebellar neurons were unstained. Control animals and 25 mg/kg animals showed an uninterrupted monolayer of Purkinje cell bodies throughout the cerebellar cortex, with densely stained dendrites that extended continuously throughout the molecular layer (Fig. 2A). Scattered loss of calbindin staining, about the width of a single Purkinje neuron, found in the cerebellar sections of 50 mg/kg animals indicated the occasional loss of single Purkinje cells (Fig 2B). With 75 or 100 mg IBO/kg, heavier Purkinje-cell loss was manifest as multiple pale, radial bands that were unstained by the anti-calbindin antisera (Figs. 2C and 2D). The bands of missing calbindin-staining following 100 mg/kg (Fig. 2D) were wider and more continuous than with 75-mg/kg doses of IBO (Fig. 2C).
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Anti-GFAP Immunocytochemistry
The increased expression of GFAP in astrocytes is classically associated with many types of neuronal injury (Aschner, 1998
; Eng and Ghirnikar, 1994; Graeber et al, 1986; O'Callaghan, 1991). The anti-GFAP immunocytochemical method provided a strong positive staining for Bergmann astrocytes and their fibers in the cerebellum (Fig. 3). Two of 6 animals exhibited narrow dark bands of GFAP positive immunoreactivity after 50 mg IBO/kg (Fig. 3B), while none of the rats that received 25 mg/kg IBO/kg (Fig. 3A) showed any distinguishable differences compared to controls. All 6 animals that were dosed with 75 or 100 mg IBO/kg showed wider, intensively GFAP-positive patches (Figs. 3C and 3D). The intensely GFAP-positive bands in the IBO-treated cerebellum occurred anatomically close to the areas of N-E-labeled neuronal degeneration and in the regions where calbindin immunoreactivity was lost. The GFAP staining method, via monitoring the intensity of GFAP protein expression in reactive astrocytes, further confirms the degeneration pattern observed with the other histochemical stain methods.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While most investigations emphasized the lesions of the cerebellar vermis (O'Hearn et al, 1993
; Scallet et al, 1996b), Molinari et al. (1996) reported additional ibogaine-induced damage to the simplex lobule, paravermis area, vermis area, and the crus-1 lobule of their 100-mg/kg rats. Our present study confirms damage to these regions of the cerebellum at doses as low as 75 mg/kg. The neurons of these areas have been reported to project axons to terminations in the deep cerebellar nuclei (as discussed by Scallet et al., 1996a,b).
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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The calbindin immunohistochemical staining provided further confirmation of the Purkinje cell degeneration. This marker, present in the cytoplasm of the Purkinje cell bodies and dendrites, appeared to vanish where the neurons were damaged, revealing such sites as unstained, white patches among the heavily labeled intact Purkinje neurons in the cerebellar cortex (Fig. 2
). The results using anti-calbindin echoed those findings with the N-E silver stain: degeneration of the Purkinje cell bodies and their dendrites as a result of ibogaine exposure (see Table 1). The advantage of the Calbindin-staining method is that it is Purkinje-neuron specific. However, a loss of calbindindin content of the neurons for any reason, including poor fixation, pharmacological down-regulation of calbindin synthesis, etc. can all be confused with a loss due to neuronal necrosis. Thus the technique should be considered as supplementary to the other approaches for neurotoxicity screening in the cerebellum.
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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1 To whom correspondence should be addressed at the Division of Neurotoxicology, National Center for Toxicological Research, 3900 NCTR Drive, Jefferson, Arkansas 72079. Fax: (870) 543-7745. E-mail: ascallet{at}nctr.fda.gov.
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