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Long-term nicotine exposure reduces Purkinje cell number in the adult rat cerebellar vermis
Neurotoxicology and Teratology 25 (2003) 329 – 334 www.elsevier.com/locate/neutera
Long-term nicotine exposure reduces Purkinje cell number in the adult rat cerebellar vermis Wei-Jung A. Chen*, Russell B. Edwards, Roland D. Romero, Scott E. Parnell, Rebecca J. Monk Department of Human Anatomy and Medical Neurobiology, College of Medicine, The Texas A&M University System Health Science Center, 240 Reynolds Medical Building, College Station, TX 77843-1114, USA Received 9 May 2002; received in revised form 27 August 2002; accepted 14 November 2002
Abstract Nicotine affects functions of the central nervous system. A previous study showed that developing cerebellar Purkinje cells are targets for early postnatal nicotine exposure. In this study, we assessed the effects of long-term nicotine exposure on mature cerebellar Purkinje cells. This is particularly relevant since the majority of smokers are exposed to nicotine over a long period. Female adult Sprague – Dawley rats received three doses of nicotine (0.01%, 0.03%, or 0.06%) through their sole water source. After 8 weeks of nicotine exposure, the cerebellar vermis was removed and processed for stereological cell counting. The results showed that this long-term nicotine treatment did not change the cerebellum weight or the size (volume) of the cerebellar vermis. The long-term nicotine treatment regimen did result in a significant loss of mature Purkinje cells in the cerebellum, however, such a loss of Purkinje cells was not nicotine dose-related. These findings indicated that the mature adult cerebellum is susceptible to the damaging effects of nicotine in depleting Purkinje cells in the cerebellum. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cerebellum; Neurotoxicity; Smoking; Stereology; Tobacco
1. Introduction Cigarette smoking produces hundreds of compounds that are readily absorbed into the physiological system of the smoker. For example, each cigarette will deliver 20 to 40 ng of benzo[a]pyrene, a compound that is suspected to be ‘‘one of the most potent mutagens and carcinogens known’’ [9] and various quantities of nicotine, a known psychoactive substance that has been suggested to be responsible for the development of nicotine dependence among smokers. Therefore, while smokers light up a cigarette, they are simultaneously exposed to many toxic agents that may produce a wide spectrum of damage to various tissue organ systems. Similarly, the use of tobacco products through other routes, such as oral (chewing tobacco), may also represent a health concern to tobacco users. One major action of nicotine is to interact with nicotinic receptors, a subpopulation of the acetylcholine receptors
* Corresponding author. Tel.: +1-979-845-4982; fax: +1-979-8450790. E-mail address: [email protected] (W.-J.A. Chen).
[14]. The pharmacological and behavioral consequences of such a cellular action and its interactions with other neurochemical systems, such as the dopaminergic system, are believed, at least in part, to be involved in the development of nicotine addiction [2,10,20]. Much of the attention of nicotine research is centered on this addiction issue, and less focus is placed on the potential toxic effects of nicotine, particularly in the area of neurotoxicology. One of our recent studies indicated that nicotine is harmful to the developing cerebellum as identified by the loss of Purkinje cells [6]. In this study, we assessed whether long-term nicotine exposure would result in the loss of mature cerebellar Purkinje cells in an adult rat model system. At present, the information available with regard to the clinical correlation linking chronic tobacco use with the cerebellar dysfunctions is limited. However, nicotine is reported to cause postural imbalance in nonsmokers and occasional smokers showing that nicotine may be affecting a circuitry involving the cerebellum [19]. Evidence from the literature further showed that the cerebellar Purkinje cells express nicotinic acetylcholine receptors, including the a4 and a7 subunits. These findings support the notion that the cerebellar Pur-
0892-0362/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0892-0362(02)00350-1
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kinje cells are the targets for nicotine and that the function of these neurons may be modified by nicotine. Therefore, we hypothesized that the number of mature Purkinje cells would be reduced following a long-term nicotine exposure regimen in a manner similar to the nicotine-induced developing Purkinje cell loss. The rationale for selecting the longterm exposure regimen and the Purkinje cells was based on the fact that most smokers are exposed to nicotine for a long period and the cerebellum is a vulnerable target for neurotoxins [11].
2. Methods 2.1. Subjects Twenty-seven adult female Sprague – Dawley rats were used in this study. They were obtained from Harlan (Houston, TX) around 90 days of age, and were housed singly in an AAALAC-accredited vivarium located at the Texas A&M University System Health Science Center. The animals were handled for a week upon arrival to accustom them to the experimenters. The experimental procedures implemented in this study were approved by University Laboratory Animal Care Committee. 2.2. Experimental procedures The animals were assigned randomly to four treatment groups with respect to the different concentrations of nicotine-containing water (0%, 0.01%, 0.03%, and 0.06%). Nicotine-containing water bottles were covered with aluminum foil due to the light-sensitive nature of the nicotine. The nicotine was administered via their sole water source, and the food was ad lib. This oral administration of nicotine is similar to the ‘‘chewing tobacco’’ or the ‘‘nicotine gum’’ route of exposure in humans, and it has been used to administer nicotine in a long-term treatment regimen in experimental research [12,17,18,21]. Water consumption and body weights were reported every third day for the first 4 weeks of treatment. The volume of daily water consumed was used to determine the actual dose of nicotine intake (mg nicotine/kg body weight). After the first 4 weeks of treatment, all animals (including the 0% control animals) were subjected to one intragastric intubation of alcohol (4 g/ kg in 22.5% w/v concentration) as part of a different ongoing study of long-term nicotine and blood alcohol concentration (results not presented). During the second 4 weeks of treatment, the water intake and body weights were monitored every 9 or 10 days since the body weights of nicotine-treated animals were catching up with that of the control animals after 4 weeks of treatment. At the end of the 8 weeks of nicotine treatment, all animals once again were given an intragastric intubation of alcohol. The animals were sacrificed 2 days following the last alcohol administration. Their brains were removed, and the cerebellar
vermes were dissected and processed for stereological cell counting. 2.3. Brain measures and tissue preparation On the day of perfusion, animals were weighed, given an overdose of Nembutal (Abbott Labs, N. Chicago) and perfused intracardially with saline followed by 4% (w/v) paraformaldehyde (in 0.1 M phosphate buffer, pH = 7.4). The cerebellum was removed, weighed, and postfixed in the same fixative until tissue preparation for stereological cell counts. The cerebellar hemispheres were removed and the central portion of the cerebellum containing the vermis was then cut sagittally into two slabs. Each slab was then dehydrated through increasing concentrations of alcohol (70%, 95%, and 100%), processed through a series of graded concentrations of infiltration solution (25%, 50%, 75%, and 100%; 100% infiltration solution is prepared by mixing 50 ml hydroxyethylmethacrylate and 0.5 g benzol peroxide; Historesin2 Embedding kit, Leica) and embedded in Historesin (15 ml of 100% infiltration solution and 1 ml dimethyl sulfoxide). Serial sectioning of each slab was set at 30 mm in the sagittal plane with a Reichert – Jung rotary microtome (Model # 2055, Leica) using a tungsten carbide knife. Every single section was saved, mounted onto glass slides, dried overnight at 60 C, stained with cresyl violet, and coverslipped. In this study, the criterion for determining whether a section was considered a vermal section was the visualization of all 10 cerebellar lobules (not necessarily 10 complete cerebellar lobules). Due to the sectioning error, one cerebellar vermis (from 0.01% NIC group) did not meet this criterion and was not included in the counting. 2.4. Verification of section thickness The thickness of the sections was verified by measuring each slab before and after sectioning, and then dividing by the total number of sections cut from that slab. The average section thickness from all slabs was 28.38 mm, which closely approached the target thickness of 30 mm. The average section thickness was used in the calculation of the reference volume. 2.5. Stereological equipment The optical disector was utilized to estimate the density of cerebellar Purkinje cells and the reference volume of cerebellum. The Nikon Optiphot microscope used in this study had a motor-driven stage [X, Y] and the adjacent attached microcator measured the Z-axis. Slides were viewed with an oil-immersion 60 objective lens with a 1.4 numerical aperture. The image from the microscope was transferred to Amiga 2000 computer using a SONY CCDIRIS color video camera. The software GRID (GRID software package, Medicosoft, Copenhagen, Denmark) pro-
W.-J.A. Chen et al. / Neurotoxicology and Teratology 25 (2003) 329–334
vided the point counting and counting frame templates, as well as their respective areas. 2.6. Stereological methods The total number of cerebellar Purkinje cells (N) was estimated from the measurement of reference volume (Vref) and numerical density (Nv) of the cells within the Vref. N ¼ Vref Nv
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P < .001] and nicotine intake [ F(2,17)= 61.61, P < .001]. However, there was no significant main effect of treatment on the body weight analysis (Fig. 1). With regard to the amount of water consumed, the control animals drank a fairly constant amount of water throughout the 8 weeks of treatment. However, water intake for all nicotine-treated groups was reduced following the beginning of nicotine exposure. However, the mean water intake for the 0.01% and 0.03% NIC groups gradually surpassed the water consumed in the control group beginning at the fourth week of treatment.
The Vref was determined by point counting and applying Cavalieri’s Principle [13], and the Purkinje cell density was determined following the optical disector method [13,30]. Details regarding the stereological methods used in the present study have been described previously [4]. 2.7. Precision of the estimates The appropriateness of the sampling scheme in its precision of the estimates was evaluated by the calculation of the coefficient of error (CE) [27,30]. The average CEs for the number of Purkinje cell nucleoli sampled for the four treatment groups ranged from 0.060 to 0.088, which were below the recommended limit of 0.10 [30]. 2.8. Statistical analysis The analyses for the body weight, water and nicotine intake were mixed analyses of variance (ANOVAs) with treatment as a between-factor and day as a within-factor. However, the cerebellum weight, the reference volume of the cerebellar vermis, the estimated number of the Purkinje cells, and the density of the Purkinje cell were analyzed by one-way ANOVAs with treatment as the between-variable. Following a significant main effect of treatment, Fisher’s least significance difference (LSD) was used for the post hoc analyses. The a level was set at .05 for all analyses.
3. Results 3.1. Body weight, water and nicotine intake Mixed ANOVAs performed on body weight, water and nicotine intake data showed significant main effects of day [ F(13,299) = 97.06, F(12,276) = 57.77, and F(12,204) = 61.61, P’s < .001, for body weight, water and nicotine intake, respectively], and interactions of treatment and day [ F(39,299) = 4.72, F(36,276) = 5.33, and F(24,204) = 11.49, P’s < .001 for body weight, water and nicotine intake, respectively]. The ANOVAs showed significant main effects of treatment on the water [ F(3,23) = 21.59,
Fig. 1. The mean body weight (A), water intake (B), and nicotine administered (C) as a function of nicotine treatment and treatment day. The vertical line on each datum point represents the standard error of the mean (S.E.M.) (n = 7, 7, 7, and 6 for 0%, 0.01%, 0.03%, and 0.06% NIC groups, respectively).
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Fig. 2. The photomicrographs of cerebellar Purkinje cells. (A) Control treatment. (B) 0.06% Nicotine treatment. Both photomicrographs were taken from lobule 4 near the mid-sagittal plane. Each arrowhead on the photomicrographs indicates a Purkinje cell, and the three arrows on the left of the photomicrograph A are used to indicate the layers of the cerebellar cortex (GL: granular cell layer; PL: Purkinje cell layer; ML: molecular layer). The length of the bar represents 25 mm for both photomicrographs A and B.
The total nicotine consumed was calculated based on the nicotine concentration of the water, the amount of nicotinecontaining water consumed, and the body weight (kg). The 0.01% NIC group consumed an average of 1 –1.5 mg/kg/ day throughout the entire 8 weeks of treatment. The nicotine consumed in the 0.03% and 0.06% NIC groups increased gradually for the first 2 weeks, and maintained at a constant level for the last 4 weeks (4 –5 and 7– 8 mg/kg/day for 0.03% and 0.06% NIC groups, respectively).
3.2. Cerebellum weight, estimated neuronal number, reference volume, and density One-way ANOVAs revealed that long-term nicotine treatment affected neither the weight of the cerebellum (Fig. 3A) nor the reference volume of the cerebellum (Fig. 3C). However, the total number of Purkinje cells and the density of the Purkinje cells (cell/mm3) in the cerebellar vermis were significantly reduced in all three nicotine-
Fig. 3. The mean cerebellum weight (A), total Purkinje cell number in the cerebellar vermis (B), the reference volume of the cerebellar vermis (C), and the density of the Purkinje cell within the cerebellar vermis (D) as a function of nicotine treatment (0%, 0.01%, 0.03%, 0.06% NIC). The asterisk indicates significant difference when compared with the control, 0% NIC group ( P < .05). Each vertical line represents the standard error of the mean (S.E.M.) (n = 7, 6, 7, and 6 for 0%, 0.01%, 0.03%, and 0.06% NIC groups, respectively).
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treated groups compared with those of the control group [Number: F(3,22) = 6.91 and Density: F(3,22) = 5.21, P’s < .01] (Figs. 2 and 3B,D). The percentage reduction in the total Purkinje cell number were 17.8%, 27.8%, and 24.1% for the 0.01%, 0.03%, and 0.06% nicotine-treated groups compared with that of the control value. There were no differences among the nicotine dose groups demonstrating that the reduction in long-term nicotine-induced Purkinje cell loss is not dose-related.
4. Discussion The findings of this study showed that long-term nicotine treatment results in a significant loss of mature cerebellar Purkinje cells in the vermal region of the adult rat cerebellum. However, the whole cerebellum weight and the volume of the cerebellar vermis did not change in response to the long-term nicotine treatment. These findings suggest that the mature Purkinje cells are sensitive to the damaging effects of nicotine indicated by the loss of cells, which is similar to the effects of nicotine reported on the developing Purkinje cells [10]. Understanding the mechanisms of how nicotine depletes Purkinje cells is beyond the scope of this research, nevertheless, it is reasonable to speculate that the interaction of nicotine and the a subunits of the nicotinic ACh receptors on the Purkinje cells may subsequently trigger the apoptotic process that leads to the loss of Purkinje cells. A recent report indicated that the activation of nicotinic receptors by low doses of nicotine results in apoptotic cell death in primary hippocampal progenitor cells [3] demonstrating the ability of nicotine in mediating apoptosis. Taken together, these findings suggest that nicotine is a neurotoxic agent regardless of its protective property in many other experimental manipulations [8, 22,32]. No significant differences in the total number of Purkinje cells were found among all three nicotine doses used in this study possibly due to a threshold level for nicotine to exert its effect on Purkinje cell loss. The blood nicotine level was not measured in this study, which is a major limitation in determining the level of blood nicotine level that is needed to mediate the Purkinje cell loss following a long period of exposure. The lack of nicotine dose-related decrease in Purkinje cell number might also be a function of the differential vulnerability of different Purkinje cell subsets to nicotine treatment. Recent evidence indicated that the cerebellar Purkinje cells are not homogeneous in their cellular structural compositions [1,5]. Therefore, it is reasonable to speculate that a subpopulation of the cerebellar Purkinje cells are sensitive to nicotine, with another subset of the Purkinje cells that are resistant to long-term nicotine treatment. Nevertheless, further investigation is needed to verify this speculation. Based on the current experimental paradigm, two alcohol intubations were given to the animals during the course of
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nicotine treatment. Although the 0% NIC control group received alcohol treatment in a similar fashion as those NIC groups, it is possible that these two alcohol doses may interact with nicotine to produce interactive effects on the Purkinje cell loss observed in this study. However, both experimental and clinical evidence suggest that nicotine may lower the peak blood alcohol concentration [7,25], the most critical determinant for alcohol-induced toxicity [28]. Therefore, should two alcohol intubations lead to a significant cell loss, the magnitude of such a cell loss would have been reduced at the presence of nicotine. In other words, the differences in Purkinje cell number observed between the 0% NIC and other NIC doses would have been greater since nicotine might protect against alcohol-mediated cell loss while at the same time exerting its toxicity in all the NIC-treated groups. The lack of change in the volume of the cerebellar vermis accompanied by the decrease in Purkinje cell number within that volume raises an important question of whether the lack of change in gross brain size (volume) is a reliable and accurate indication of an intact, normally functioning brain. A recent paper by West et al. [29] brings up the same concern in the field of fetal alcohol research. The magnetic resonance imaging (MRI) has been a popular technique to assess brain anomalies in fetal alcohol syndrome patients [15,16,24,26]. In general, the MRI findings are interpreted as negative if no change in any specific brain region is found, i.e. no change in volume indicates no injury to that region resulting from the neurotoxic insult. However, the current results and the findings from West et al. [29] group validate the need to use more sensitive microscopic assessments in animal studies to confirm the clinical observations using MRI techniques. On the other hand, the gross measurements from MRI techniques remain one of the most powerful and valuable methods to detect morphological pathology in living organisms, but the lack of pathology in certain anatomical structures should be interpreted cautiously. Despite the negative effects of long-term nicotine treatment, it needs to be recognized that there are some reported beneficial effects of chronic nicotine exposure. Levin et al. [23,31] reported that chronic nicotine treatment improved cognitive performance both in human and animal studies (nicotine skin patch for humans and osmotic minipump for Sprague – Dawley rats). However, no improvement in motor function was evident in patients with Alzheimer’s disease. Furthermore, it should be noted that the enhancing effect in performance dissipated following the removal of the nicotine suggesting that the presence of the nicotine in the physiological system is required to exert such facilitating effects. However, what is lacking in the literature is whether the decremental performance of those long-term nicotineexposed patients following the cessation of the treatment is a function of the long-term nicotine treatment, the withdrawal of the nicotine, or both. In conclusion, this study showed that long-term nicotine exposure during adulthood resulted in the loss of the
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cerebellar Purkinje cells in a rat model system. At present, the significance and manifestations of such a loss of mature Purkinje cells remain to be determined. Future behavioral research related to cerebellar functions in the area of neurotoxicity of nicotine will shed some light on this issue.
Acknowledgements This research was supported by NIH grant NS39899 to W.-J.A.C.
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Long-term nicotine exposure reduces Purkinje cell number in the adult rat cerebellar vermis Wei-Jung A. Chen*, Russell B. Edwards, Roland D. Romero, Scott E. Parnell, Rebecca J. Monk Department of Human Anatomy and Medical Neurobiology, College of Medicine, The Texas A&M University System Health Science Center, 240 Reynolds Medical Building, College Station, TX 77843-1114, USA Received 9 May 2002; received in revised form 27 August 2002; accepted 14 November 2002
Abstract Nicotine affects functions of the central nervous system. A previous study showed that developing cerebellar Purkinje cells are targets for early postnatal nicotine exposure. In this study, we assessed the effects of long-term nicotine exposure on mature cerebellar Purkinje cells. This is particularly relevant since the majority of smokers are exposed to nicotine over a long period. Female adult Sprague – Dawley rats received three doses of nicotine (0.01%, 0.03%, or 0.06%) through their sole water source. After 8 weeks of nicotine exposure, the cerebellar vermis was removed and processed for stereological cell counting. The results showed that this long-term nicotine treatment did not change the cerebellum weight or the size (volume) of the cerebellar vermis. The long-term nicotine treatment regimen did result in a significant loss of mature Purkinje cells in the cerebellum, however, such a loss of Purkinje cells was not nicotine dose-related. These findings indicated that the mature adult cerebellum is susceptible to the damaging effects of nicotine in depleting Purkinje cells in the cerebellum. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cerebellum; Neurotoxicity; Smoking; Stereology; Tobacco
1. Introduction Cigarette smoking produces hundreds of compounds that are readily absorbed into the physiological system of the smoker. For example, each cigarette will deliver 20 to 40 ng of benzo[a]pyrene, a compound that is suspected to be ‘‘one of the most potent mutagens and carcinogens known’’ [9] and various quantities of nicotine, a known psychoactive substance that has been suggested to be responsible for the development of nicotine dependence among smokers. Therefore, while smokers light up a cigarette, they are simultaneously exposed to many toxic agents that may produce a wide spectrum of damage to various tissue organ systems. Similarly, the use of tobacco products through other routes, such as oral (chewing tobacco), may also represent a health concern to tobacco users. One major action of nicotine is to interact with nicotinic receptors, a subpopulation of the acetylcholine receptors
* Corresponding author. Tel.: +1-979-845-4982; fax: +1-979-8450790. E-mail address: [email protected] (W.-J.A. Chen).
[14]. The pharmacological and behavioral consequences of such a cellular action and its interactions with other neurochemical systems, such as the dopaminergic system, are believed, at least in part, to be involved in the development of nicotine addiction [2,10,20]. Much of the attention of nicotine research is centered on this addiction issue, and less focus is placed on the potential toxic effects of nicotine, particularly in the area of neurotoxicology. One of our recent studies indicated that nicotine is harmful to the developing cerebellum as identified by the loss of Purkinje cells [6]. In this study, we assessed whether long-term nicotine exposure would result in the loss of mature cerebellar Purkinje cells in an adult rat model system. At present, the information available with regard to the clinical correlation linking chronic tobacco use with the cerebellar dysfunctions is limited. However, nicotine is reported to cause postural imbalance in nonsmokers and occasional smokers showing that nicotine may be affecting a circuitry involving the cerebellum [19]. Evidence from the literature further showed that the cerebellar Purkinje cells express nicotinic acetylcholine receptors, including the a4 and a7 subunits. These findings support the notion that the cerebellar Pur-
0892-0362/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0892-0362(02)00350-1
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kinje cells are the targets for nicotine and that the function of these neurons may be modified by nicotine. Therefore, we hypothesized that the number of mature Purkinje cells would be reduced following a long-term nicotine exposure regimen in a manner similar to the nicotine-induced developing Purkinje cell loss. The rationale for selecting the longterm exposure regimen and the Purkinje cells was based on the fact that most smokers are exposed to nicotine for a long period and the cerebellum is a vulnerable target for neurotoxins [11].
2. Methods 2.1. Subjects Twenty-seven adult female Sprague – Dawley rats were used in this study. They were obtained from Harlan (Houston, TX) around 90 days of age, and were housed singly in an AAALAC-accredited vivarium located at the Texas A&M University System Health Science Center. The animals were handled for a week upon arrival to accustom them to the experimenters. The experimental procedures implemented in this study were approved by University Laboratory Animal Care Committee. 2.2. Experimental procedures The animals were assigned randomly to four treatment groups with respect to the different concentrations of nicotine-containing water (0%, 0.01%, 0.03%, and 0.06%). Nicotine-containing water bottles were covered with aluminum foil due to the light-sensitive nature of the nicotine. The nicotine was administered via their sole water source, and the food was ad lib. This oral administration of nicotine is similar to the ‘‘chewing tobacco’’ or the ‘‘nicotine gum’’ route of exposure in humans, and it has been used to administer nicotine in a long-term treatment regimen in experimental research [12,17,18,21]. Water consumption and body weights were reported every third day for the first 4 weeks of treatment. The volume of daily water consumed was used to determine the actual dose of nicotine intake (mg nicotine/kg body weight). After the first 4 weeks of treatment, all animals (including the 0% control animals) were subjected to one intragastric intubation of alcohol (4 g/ kg in 22.5% w/v concentration) as part of a different ongoing study of long-term nicotine and blood alcohol concentration (results not presented). During the second 4 weeks of treatment, the water intake and body weights were monitored every 9 or 10 days since the body weights of nicotine-treated animals were catching up with that of the control animals after 4 weeks of treatment. At the end of the 8 weeks of nicotine treatment, all animals once again were given an intragastric intubation of alcohol. The animals were sacrificed 2 days following the last alcohol administration. Their brains were removed, and the cerebellar
vermes were dissected and processed for stereological cell counting. 2.3. Brain measures and tissue preparation On the day of perfusion, animals were weighed, given an overdose of Nembutal (Abbott Labs, N. Chicago) and perfused intracardially with saline followed by 4% (w/v) paraformaldehyde (in 0.1 M phosphate buffer, pH = 7.4). The cerebellum was removed, weighed, and postfixed in the same fixative until tissue preparation for stereological cell counts. The cerebellar hemispheres were removed and the central portion of the cerebellum containing the vermis was then cut sagittally into two slabs. Each slab was then dehydrated through increasing concentrations of alcohol (70%, 95%, and 100%), processed through a series of graded concentrations of infiltration solution (25%, 50%, 75%, and 100%; 100% infiltration solution is prepared by mixing 50 ml hydroxyethylmethacrylate and 0.5 g benzol peroxide; Historesin2 Embedding kit, Leica) and embedded in Historesin (15 ml of 100% infiltration solution and 1 ml dimethyl sulfoxide). Serial sectioning of each slab was set at 30 mm in the sagittal plane with a Reichert – Jung rotary microtome (Model # 2055, Leica) using a tungsten carbide knife. Every single section was saved, mounted onto glass slides, dried overnight at 60 C, stained with cresyl violet, and coverslipped. In this study, the criterion for determining whether a section was considered a vermal section was the visualization of all 10 cerebellar lobules (not necessarily 10 complete cerebellar lobules). Due to the sectioning error, one cerebellar vermis (from 0.01% NIC group) did not meet this criterion and was not included in the counting. 2.4. Verification of section thickness The thickness of the sections was verified by measuring each slab before and after sectioning, and then dividing by the total number of sections cut from that slab. The average section thickness from all slabs was 28.38 mm, which closely approached the target thickness of 30 mm. The average section thickness was used in the calculation of the reference volume. 2.5. Stereological equipment The optical disector was utilized to estimate the density of cerebellar Purkinje cells and the reference volume of cerebellum. The Nikon Optiphot microscope used in this study had a motor-driven stage [X, Y] and the adjacent attached microcator measured the Z-axis. Slides were viewed with an oil-immersion 60 objective lens with a 1.4 numerical aperture. The image from the microscope was transferred to Amiga 2000 computer using a SONY CCDIRIS color video camera. The software GRID (GRID software package, Medicosoft, Copenhagen, Denmark) pro-
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vided the point counting and counting frame templates, as well as their respective areas. 2.6. Stereological methods The total number of cerebellar Purkinje cells (N) was estimated from the measurement of reference volume (Vref) and numerical density (Nv) of the cells within the Vref. N ¼ Vref Nv
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P < .001] and nicotine intake [ F(2,17)= 61.61, P < .001]. However, there was no significant main effect of treatment on the body weight analysis (Fig. 1). With regard to the amount of water consumed, the control animals drank a fairly constant amount of water throughout the 8 weeks of treatment. However, water intake for all nicotine-treated groups was reduced following the beginning of nicotine exposure. However, the mean water intake for the 0.01% and 0.03% NIC groups gradually surpassed the water consumed in the control group beginning at the fourth week of treatment.
The Vref was determined by point counting and applying Cavalieri’s Principle [13], and the Purkinje cell density was determined following the optical disector method [13,30]. Details regarding the stereological methods used in the present study have been described previously [4]. 2.7. Precision of the estimates The appropriateness of the sampling scheme in its precision of the estimates was evaluated by the calculation of the coefficient of error (CE) [27,30]. The average CEs for the number of Purkinje cell nucleoli sampled for the four treatment groups ranged from 0.060 to 0.088, which were below the recommended limit of 0.10 [30]. 2.8. Statistical analysis The analyses for the body weight, water and nicotine intake were mixed analyses of variance (ANOVAs) with treatment as a between-factor and day as a within-factor. However, the cerebellum weight, the reference volume of the cerebellar vermis, the estimated number of the Purkinje cells, and the density of the Purkinje cell were analyzed by one-way ANOVAs with treatment as the between-variable. Following a significant main effect of treatment, Fisher’s least significance difference (LSD) was used for the post hoc analyses. The a level was set at .05 for all analyses.
3. Results 3.1. Body weight, water and nicotine intake Mixed ANOVAs performed on body weight, water and nicotine intake data showed significant main effects of day [ F(13,299) = 97.06, F(12,276) = 57.77, and F(12,204) = 61.61, P’s < .001, for body weight, water and nicotine intake, respectively], and interactions of treatment and day [ F(39,299) = 4.72, F(36,276) = 5.33, and F(24,204) = 11.49, P’s < .001 for body weight, water and nicotine intake, respectively]. The ANOVAs showed significant main effects of treatment on the water [ F(3,23) = 21.59,
Fig. 1. The mean body weight (A), water intake (B), and nicotine administered (C) as a function of nicotine treatment and treatment day. The vertical line on each datum point represents the standard error of the mean (S.E.M.) (n = 7, 7, 7, and 6 for 0%, 0.01%, 0.03%, and 0.06% NIC groups, respectively).
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Fig. 2. The photomicrographs of cerebellar Purkinje cells. (A) Control treatment. (B) 0.06% Nicotine treatment. Both photomicrographs were taken from lobule 4 near the mid-sagittal plane. Each arrowhead on the photomicrographs indicates a Purkinje cell, and the three arrows on the left of the photomicrograph A are used to indicate the layers of the cerebellar cortex (GL: granular cell layer; PL: Purkinje cell layer; ML: molecular layer). The length of the bar represents 25 mm for both photomicrographs A and B.
The total nicotine consumed was calculated based on the nicotine concentration of the water, the amount of nicotinecontaining water consumed, and the body weight (kg). The 0.01% NIC group consumed an average of 1 –1.5 mg/kg/ day throughout the entire 8 weeks of treatment. The nicotine consumed in the 0.03% and 0.06% NIC groups increased gradually for the first 2 weeks, and maintained at a constant level for the last 4 weeks (4 –5 and 7– 8 mg/kg/day for 0.03% and 0.06% NIC groups, respectively).
3.2. Cerebellum weight, estimated neuronal number, reference volume, and density One-way ANOVAs revealed that long-term nicotine treatment affected neither the weight of the cerebellum (Fig. 3A) nor the reference volume of the cerebellum (Fig. 3C). However, the total number of Purkinje cells and the density of the Purkinje cells (cell/mm3) in the cerebellar vermis were significantly reduced in all three nicotine-
Fig. 3. The mean cerebellum weight (A), total Purkinje cell number in the cerebellar vermis (B), the reference volume of the cerebellar vermis (C), and the density of the Purkinje cell within the cerebellar vermis (D) as a function of nicotine treatment (0%, 0.01%, 0.03%, 0.06% NIC). The asterisk indicates significant difference when compared with the control, 0% NIC group ( P < .05). Each vertical line represents the standard error of the mean (S.E.M.) (n = 7, 6, 7, and 6 for 0%, 0.01%, 0.03%, and 0.06% NIC groups, respectively).
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treated groups compared with those of the control group [Number: F(3,22) = 6.91 and Density: F(3,22) = 5.21, P’s < .01] (Figs. 2 and 3B,D). The percentage reduction in the total Purkinje cell number were 17.8%, 27.8%, and 24.1% for the 0.01%, 0.03%, and 0.06% nicotine-treated groups compared with that of the control value. There were no differences among the nicotine dose groups demonstrating that the reduction in long-term nicotine-induced Purkinje cell loss is not dose-related.
4. Discussion The findings of this study showed that long-term nicotine treatment results in a significant loss of mature cerebellar Purkinje cells in the vermal region of the adult rat cerebellum. However, the whole cerebellum weight and the volume of the cerebellar vermis did not change in response to the long-term nicotine treatment. These findings suggest that the mature Purkinje cells are sensitive to the damaging effects of nicotine indicated by the loss of cells, which is similar to the effects of nicotine reported on the developing Purkinje cells [10]. Understanding the mechanisms of how nicotine depletes Purkinje cells is beyond the scope of this research, nevertheless, it is reasonable to speculate that the interaction of nicotine and the a subunits of the nicotinic ACh receptors on the Purkinje cells may subsequently trigger the apoptotic process that leads to the loss of Purkinje cells. A recent report indicated that the activation of nicotinic receptors by low doses of nicotine results in apoptotic cell death in primary hippocampal progenitor cells [3] demonstrating the ability of nicotine in mediating apoptosis. Taken together, these findings suggest that nicotine is a neurotoxic agent regardless of its protective property in many other experimental manipulations [8, 22,32]. No significant differences in the total number of Purkinje cells were found among all three nicotine doses used in this study possibly due to a threshold level for nicotine to exert its effect on Purkinje cell loss. The blood nicotine level was not measured in this study, which is a major limitation in determining the level of blood nicotine level that is needed to mediate the Purkinje cell loss following a long period of exposure. The lack of nicotine dose-related decrease in Purkinje cell number might also be a function of the differential vulnerability of different Purkinje cell subsets to nicotine treatment. Recent evidence indicated that the cerebellar Purkinje cells are not homogeneous in their cellular structural compositions [1,5]. Therefore, it is reasonable to speculate that a subpopulation of the cerebellar Purkinje cells are sensitive to nicotine, with another subset of the Purkinje cells that are resistant to long-term nicotine treatment. Nevertheless, further investigation is needed to verify this speculation. Based on the current experimental paradigm, two alcohol intubations were given to the animals during the course of
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nicotine treatment. Although the 0% NIC control group received alcohol treatment in a similar fashion as those NIC groups, it is possible that these two alcohol doses may interact with nicotine to produce interactive effects on the Purkinje cell loss observed in this study. However, both experimental and clinical evidence suggest that nicotine may lower the peak blood alcohol concentration [7,25], the most critical determinant for alcohol-induced toxicity [28]. Therefore, should two alcohol intubations lead to a significant cell loss, the magnitude of such a cell loss would have been reduced at the presence of nicotine. In other words, the differences in Purkinje cell number observed between the 0% NIC and other NIC doses would have been greater since nicotine might protect against alcohol-mediated cell loss while at the same time exerting its toxicity in all the NIC-treated groups. The lack of change in the volume of the cerebellar vermis accompanied by the decrease in Purkinje cell number within that volume raises an important question of whether the lack of change in gross brain size (volume) is a reliable and accurate indication of an intact, normally functioning brain. A recent paper by West et al. [29] brings up the same concern in the field of fetal alcohol research. The magnetic resonance imaging (MRI) has been a popular technique to assess brain anomalies in fetal alcohol syndrome patients [15,16,24,26]. In general, the MRI findings are interpreted as negative if no change in any specific brain region is found, i.e. no change in volume indicates no injury to that region resulting from the neurotoxic insult. However, the current results and the findings from West et al. [29] group validate the need to use more sensitive microscopic assessments in animal studies to confirm the clinical observations using MRI techniques. On the other hand, the gross measurements from MRI techniques remain one of the most powerful and valuable methods to detect morphological pathology in living organisms, but the lack of pathology in certain anatomical structures should be interpreted cautiously. Despite the negative effects of long-term nicotine treatment, it needs to be recognized that there are some reported beneficial effects of chronic nicotine exposure. Levin et al. [23,31] reported that chronic nicotine treatment improved cognitive performance both in human and animal studies (nicotine skin patch for humans and osmotic minipump for Sprague – Dawley rats). However, no improvement in motor function was evident in patients with Alzheimer’s disease. Furthermore, it should be noted that the enhancing effect in performance dissipated following the removal of the nicotine suggesting that the presence of the nicotine in the physiological system is required to exert such facilitating effects. However, what is lacking in the literature is whether the decremental performance of those long-term nicotineexposed patients following the cessation of the treatment is a function of the long-term nicotine treatment, the withdrawal of the nicotine, or both. In conclusion, this study showed that long-term nicotine exposure during adulthood resulted in the loss of the
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cerebellar Purkinje cells in a rat model system. At present, the significance and manifestations of such a loss of mature Purkinje cells remain to be determined. Future behavioral research related to cerebellar functions in the area of neurotoxicity of nicotine will shed some light on this issue.
Acknowledgements This research was supported by NIH grant NS39899 to W.-J.A.C.
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