Cell Biol Toxicol 2006; 22: 119–125. DOI: 10.1007/s10565-006-0037-9
C Springer 2006
A calcium channel blocker flunarizine attenuates the neurotoxic effects of iron ¨ M. Omer Bostanci1,∗ , Faruk Ba˘girici1 and Sinan Canan2 1 Department of Physiology, Faculty of Medicine, University of Ondokuz Mayis, 55139 Samsun, Turkey; 2 Department of Physiology, Faculty of Medicine, University of Baskent, 06530 Ankara, Turkey Received 25 August 2005; accepted 9 December 2005
Keywords: iron, hippocampus, cell death, flunarizine, stereology Abstract Iron is a metal highly concentrated in liver and brain tissue, and known to induce neuronal hyperactivity and oxidative stress. It has been established that iron levels rise in the brain in some neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases (AD). A body of evidence indicates a link between neuronal death and intracellular excessive calcium accumulation. The aim of the present study was to investigate the effects of a calcium antagonist, flunarizine, on neurotoxicity induced by intracerebroventricular (i.c.v.) iron injection. For this reason rats were divided into three groups as control, iron and iron+flunarizine groups. Animals in iron and iron+flunarizine groups received i.c.v. FeCl3 injection (200 mM, 2.5 μl), while control rats received the same amount of saline into the cerebral ventricles. Rats in iron+flunarizine group also received i.c.v. flunarizine (1 μM, 2 μl) following FeCl3 injection. All animals were kept alive for ten days following the operation and animals in iron+flunarizine group received intraperitoneal (i.p.) flunarizine injections once a day (10 mg/kg/day) during this period. After ten days, rats were sacrificed. The total numbers of neurons in hippocampus of all rats were estimated with the latest, unbiased stereological techniques. Findings of the present study suggest that flunarizine may attenuate the neurotoxic effects of iron injection by inhibiting the cellular influx of excessive calcium ions.
Introduction Iron is the most abundant metal in the human body and it is known to be more concentrated than any other metals especially in liver and brain tissue (Gerlach et al., 1994; Youdim, 1998). Distribution of iron in the brain is rather irregular and it reaches higher concentrations in basal ganglia (subtantia nigra, putamen, caudate nucleus and globus pallidus), red nucleus and dentate nucleus (Albers et al., 1992). In many neurodegenerative dis-
orders including Hallervorden-Spatz Syndrome, Parkinson’s and Alzheimer’s diseases iron has been shown to be excessively accumulated in the brain (Swaiman, 1991; Qian et al., 1997; Aisen et al., 1999). Iron accumulation was held responsible for the potentiation of oxidative stress and neuronal death (Qian and Wang, 1998; Sayre et al., 1999). Previous reports indicate that the iron concentration in amigdala, pyriform cortex, hippocampus and olfactory region elevated markedly in Alzheimer’s disease (Samudralwer
120 et al., 1995; Deibel et al., 1996). Iron is also thought to be involved in the mechanism of posttraumatic epileptogenesis (Willmore and Triggs, 1991). It has been shown both behaviorally and electrophysiologically that the administration of FeCl2 or FeCl3 into the sensorymotor cortices of experimental animals lead to epileptiform discharges (Willmore et al., 1978). Subpial injections of iron salts lead to formation of brain edema, cavity lesions and recurrent epileptiform discharges as well as inducing the free radical formation (Willmore et al., 1983). Free radicals lead to disintegration of plasma membrane by causing lipid peroxidation (Porter et al., 1995). As a result of such disintegration, ionic gradients cannot be preserved sufficiently and this eventually leads to an excessive ion influx, including calcium, into the cells (Robb et al., 1999). Intracellular calcium accumulation in neuronal cells is the main factor thought to be responsible for cellular death (Landfield et al., 1992; Beal, 1995; Dawson et al., 1995; Orrenius et al., 1996). Current literature on neurotoxicity does not contain sufficient information on the effects of calcium channel blockers on quantitative aspects of neuronal loss induced by iron. In the present study we aimed to investigate the effects of a potent calcium channel antagonist flunarizine on FeCl3 induced neuronal loss, using new, unbiased stereological techniques.
Materials and methods Animals Thirty adult male Wistar albino rats, weighing 210 ± 25 g, belonging the same age group were divided into three groups as control (n = 10), iron (n = 10) and iron+flunarizine (n = 10). All animals were obtained from Experimental Research Center of Ondokuz Mayis University. All animals were kept in constant laboratory conditions and supplied with food and water ad-libidum.
Operation Animals were kept away from food for 12 h prior to surgery and all animals were weighted just before the surgical operation. Anesthesia was induced by i.p. injection of ketamine hydrochloride (100 mg/kg). After the skin covering the skulls was shaved and animals were fixed to a stereotaxic apparatus, a rostro-caudal incision of 2 mm length was made using an electric cutter (Ellman Surgitron). After the Bregma line exposed clearly, a hole with a diameter of 1 mm was drilled at the point located 2 mm left and 0.6 mm posterior to Bregma using a dental drill. All drugs administered were given through this hole to a depth of 4.2 mm using a Hamilton microinjector. Rats in the control group received 2.5 μl saline while rats in iron group received 200 mM (2.5 μl) FeCl3 (Willmore et al., 1983). Rats in iron+flunarizine group received the same amount of FeCl3 and i.c.v. flunarizine (1 μM, 2 μl). Then, incisions were sutured and incision area was cleaned using 10% povidon iodide just prior the placement of the animals to their cages. All animals were survived for ten days following the surgery. Only the rats belonging to iron+flunarizine group received additional i.p. flunarizine treatment as 10 mg/kg/day for ten days. The first dose of flunarizine was administered during the first five minutes following the surgical operation. Rats belonging to other groups received same amount of i.p. saline injection for ten-day survival period. After the survival period, all animals perfused intracardially under deep urethane anesthesia with 10% formaldehyde and saline, buffered for pH = 7.2. After the completion of the perfusion process all animals were decapitated, brains were removed immediately and placed in the same fixative for postfixation. Brains of the three animals from each group were used for the determination of tissue iron levels. After the cerebra and cerebella were separated physically, brains were processed using the standard histological techniques and embedded in paraplast embedding media. Serial tissue sections
121 obtained using a rotary microtome (Leica RM 2135) in horizontal plane with a section thickness of 40 μm. Approval of Ethical Committee of Ondokuz Mayis University has been obtained prior to experiments and all animal work was performed according to the Experimental Animal Care Rules of European Community Council. Section sampling and the determination of total neuron number The cytoarchitectonic characteristics of the CAl, CA2 and CA3 pyramidal cell layers were identified using the criteria of West et al. (1991). Although these layers are well delineated at the central levels of the septotemporal axis of the hippocampus, delineation becomes more complicated as one proceeds towards the septal and temporal poles. In the preparations used, the characteristic features of the CA1, CA2 and CA3 layers were not found to be sufficient for consistent definition at some peripheral levels. Since unambiguous definition of profile boundaries at all levels is a prerequisite to obtain a reliable quantitative data, we considered these subdivisions as a single layer (Figure 1).
Figure 1. General view of hippocampal formation (10×). Area delineated using dashed lines is the area used for pyramidal cell counting.
According to the pilot study, 14–17 sections were sampled in a systematic-random fashion (ssf: 1/7) out of a total of 130 horizontal sections per individual hippocampi. First sections were chosen randomly from the first set of 7 sections containing the hippocampus and then the consecutive samples were selected with a fixed interval of 7 sections. Hippocampal pyramidal neurons were counted using the optical fractionator counting method which is a combination of fractionator sampling scheme and disector counting technique (West et al., 1991). All counting and analysis were performed using a modified computer-assisted stereological analysis system. Areas for cell counting were determined and delineated using CAST Grid stereological analysis software (Olympus, Denmark). Cell counts were done using a sampling scheme optimized for a total of approximately 500 cell counts per individual. Determined pyramidal sectional areas were scanned automatically using consecutive steps with 250 × 250 μm x-y size. Every step in this scanning was individually analyzed with optical disector probes using 100× oil-objectives. During optical disector application, an unbiased counting frame comprising the 15% of the total step area was used for particle sampling and counting. Thus the area sampling fraction (asf) is determined as 445 μm2 /62500 μm2 . The last sampling level in optical fractionator applications is the thickness sampling stage. Optical disector counting requires a virtual vertical scanning of the section of interest in order to count stacks of particles. According to previous pilot studies, a fixed disector height of 10 μm was predetermined and used throughout the study. This height is formed by virtual movement of a section plane (the focal plane) through the section thickness. Generally a narrow upper “guard zone” passed before the actual optic disector counting in order to avoid the possible irregularities of the sectional surface. Here we left a 5 μm upper guard zone, applied particle counting through a 10 μm disector height and measured the section thickness. All such measurements were done using a
122 digital microcator (Hidenhein, Germany), incorporated in the stereological analysis system. Thus the final sampling stage, generally called the thickness sampling fraction (tsf) was calculated by [Disector Height]/[Mean Section Thickness]. Average section thickness was estimated for each section by measuring the thickness of every 10th field of counting with a random start and by averaging the measured thickness values for each section. The total avarage of section thickness was 23.60 ± 1.74 amog all animals. After completing a throughout sampling for all sampled sections, properly sampled pyramidal cells were counted as disector particles (Q− ). Total number of hippocampal pyramidal neurons (N) was then calculated using the following formulation: N = [1/ssf] × [1/asf] × [1/tsf] ×
Q−
Results obtained from counting were checked for distribution using Kolmogorov-Smirnov Test. Hemispheric differences in terms of neuron numbers were tested using paired T-test and differences between groups were tested using Post hoc Tukey HSD test. Determination of tissue levels Iron levels of the brain tissue were determined by dry ashing method using an atomic absorption apparatus (Perkin-Elmer 2280, FLAME). Since Fe2+ and Fe3+ converted to each other, total amount of Fe2+ and Fe3+ were determined. Chemicals Flunarizine, Cresyl violet and FeCl3 6H2 O obtained from Sigma Chemical Co. (St. Louis, Mo, USA); entellan, xylene and acetic acid obtained from Merck (Darmstadt, Germany); formaldehyde, chloroform and others were obtained from Aklar Chemistry (Ankara, Turkey).
Table 1. Total pyramidal cell numbers in left and right hippocampus Regions
Groups
Total cell number (N ± SEM)
CV
CE
Left Hipp
Control Iron Iron+Flu Control Iron Iron+Flu
645119 ± 14765 310817 ± 14974 518778 ± 26908 657048 ± 26432 318624 ± 22571 532985 ± 28327
0.0339 0.0949 0.0386 0.0342 0.1071 0.0167
0.051 0.062 0.056 0.053 0.063 0.055
Right Hipp
SEM, standard error of the mean; CV, coefficient of variation; CE, coefficient of error; Flu, flunarizine; Hipp, Hippocampus.
Results Total iron levels in rat brain tissue were found to be 112 ± 10 μg, in control rats; 172 ± 13 μg in iron group and 168 ± 11 μg in iron+flunarizine group. Difference between control and iron treated groups were statistically significant ( p < 0.001). Table 1 shows the estimated mean total neuron number of left hippocampal neurons in control, iron and iron+flunarizine groups. After comparing iron group with controls, rats in iron group were appeared to have 51.8% less number of neurons than control group and this difference was statistically significant ( p < 0.001). Rats in iron+flunarizine group have 19.5% lower hippocampal cell numbers with respect to controls ( p < 0.001). Comparison between iron and iron+flunarizine rats revealed that flunarizine significantly attenuates the iron-induced neuron loss from 51.8% to 19.5% and protect hippocampal neurons against iron toxicity ( p < 0.001). Estimated mean total number of right hippocampal neurons in control, iron and iron+ flunarizine groups are listed in Table 1. Iron group display a marked decrease (51.5%) in hippocampal neuron number when compared to controls ( p < 0.001). Neuron loss in iron+flunarizine group with respect to controls was also significant and was 18.8% ( p < 0.001). Flunarizine appears to attenuate the iron-induced neuron loss from 51.5% to 18.8% and seems to have a significant
123 neuroprotective effect against the iron neurotoxicity ( p < 0.001). Discussion The present study focused on the effect of a calcium channel blocker, flunarizine, on intracerebroventricular applied iron (FeCl3 )-induced neuronal loss using the latest unbiased stereological techniques. Total number of neurons in hippocampus was determined by optical fractionator technique, which is a combination of systematic random sampling and the optical disector counting method (West et al., 1991). These methods are known to efficiently prevent sources of bias from individuals and from the methodology itself, and hence allow us to determine the number of neurons in a certain brain region in an unbiased and efficient manner (Sterio, 1984; Gundersen, 1986; Gundersen et al., 1988). In modern stereological cell counting approaches, each and every cell filling the region of interest have an equal probability of being a part of the final sample. This ensures the unbiased sampling and unbiased estimation of total neuron number. Iron as well has been used to induce epileptic activity. Meyerhoff et al. (2004) have applied iron chloride intracortically to induce epilepsy (600 mM in a volume of 10 μl). In our study, howewer, none of the animals displayed seizure behavior during the survival period. We have injected 2.5 μl of 200 mM iron chloride (i.c.v.) solution to induce free radical formation. Hence our dose of iron may not be sufficient to trigger a detectable level of epileptiform activity. In addition, the route of iron application in our experimental design might be another factor for absence of epileptic behaviour. Hippocampus is known to be involved in learning and memory pocesses. Previous studies indicate that in Alzheimer’s disease, iron levels in amygdala, pyriform cortex, hippocampus and olfactory regions were shown to be elevated significantly (Samudralwer et al., 1995; Deibel et al.,
1996). It is also known that the subpial injections of iron salt solutions results in an induction of free radical formation (Willmore et al., 1983). Iron is a frequently used metal in order to induce lipid peroxidation and cellular damage. Ferro (Fe2+ ) and ferric (Fe3+ ) iron produce hydroxyl radicals via Fenton and Haber-Weis reactions and cause cellular damage (Aust et al., 1985; Braughler et al., 1986). We have also demonstrated in previous study that intracortically administrated iron causes hippocampal neuronal loss (Bostanci et al., 2003). Free radicals are the products of routine energy metabolism and hence produced continuously during the oxidation-reduction reactions in the cell. When the concentrations of those radicals exceed the normal levels, they start to bind the unsaturated bonds of fatty acids and cholesterol, lead to membrane peroxidation and membrane disintegration (Porter et al., 1995). This disruption of membrane integrity threatens the transmembrane differences of ionic concentrations and cations, especially calcium begin to enter the cell (Robb et al., 1999). Elevated intracellular Ca2+ levels in neurons are thought to mediate the oxidative cellular death (Landfield et al., 1992; Beal, 1995; Dawson et al., 1995; Orrenius et al., 1996). Oxidative stress and excitotoxicity are two basic mechanisms leading to pathological neuronal death in the nervous system (Choi, 1988; Albers et al., 1992). Evidence suggests that these two pathological processes may be interconnected or even interwoven in neuronal damage. Excitotoxicity involves increased cellular influx of calcium ions, resulting activation of calciumdependent phospholipases and/or NO synthase and thus increased free radical formation. An increase in free radical generation also induces more glutamate release and this process also deteriorates the excitotoxic damage (PellegriniGiampietro et al., 1988; Izumi et al., 1992). Induction of oxidative stress is also known to affect the calcium concentration in mitochondria, which is an important calcium store in the cell (Chakraborti
124 et al., 1999). Excessive calcium influx into the cell also results in glutamate release, which is the major neurotransmitter in the central nervous system. Glutamate activates the ligand-gated ion channels including NMDA, kainic acid and quisqualate channels and leads to Na+ and Ca2+ influx into the postsynaptic cells. Sodium influx results in the depolarization of the cell membrane and hence leads to opening of voltage-gated calcium channels on the cell membrane. Resulting excessive calcium influx is generally held responsible for triggering of epileptic discharges and cellular death (Uematsu et al., 1990). It has been shown in our previous study using electrophysiological techniques that flunarizine had antiepileptic activity in penicillin-induced experimental epilepsy (Bagirici et al., 2001). Increased intracellular calcium level is very important in the genesis of ischemic damage (Flayn et al., 1989). Necrotic cells have a high level of intracellular calcium (Farber, 1981) and removal of calcium from extracellular space has been shown to diminish the rate of cellular death (Schanne et al., 1979; Hansen and Zeuthen, 1981). Calcium antagonists prevent calcium influx into the cell and such substances are readily used in clinical practice to prevent ischemic brain damage (Weiner, 1988). Flunarizine blocks L-type calcium channels as well as T-type channels in certain tissues (Tytgat et al., 1988). T-type calcium channels are widely distributed in the body and are more concentrated in neuronal cells (Carbone and Lux, 1984). Flunarizine has a high affinity for the membrane binding regions of nitrendipine, a dihydropiridine compound, and it has been hypothesized that flunarizine exerts its channel blocking activity through those binding sites (Leysen and Gommeron, 1984). It has been suggested that calcium channel blockers are also membrane protecting agents by preventing the peroxidation of the lipid bilayer (Herbelte and Katz, 1987). It is also known that such drugs have antioxidant effects in different brain regions (Zaleska and Floyd, 1985).
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¨ Address for correspondence: M. Omer Bostanci, Department of Physiology, Faculty of Medicine, University of Ondokuz Mayis, 55139 Samsun, Turkey. E-mail:
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