Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-015-1148-8
ORIGINAL ARTICLE
Protective effect of a calcium channel blocker “diltiazem” on aluminum chloride-induced dementia in mice Anu Rani 1 & Neha 2
&
Rupinder K. Sodhi 2 & Amanpreet Kaur 1
Received: 11 May 2015 / Accepted: 19 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Many studies report that heavy metals such as aluminum are involved in amyloid beta aggregation and neurotoxicity. Further, high concentration of aluminum in the brain deregulates calcium signaling which contributes to synaptic dysfunction and halts neuronal communication which ultimately leads to the development of Alzheimer’s disease. Recently, diltiazem, a calcium channel blocker clinically used in angina, is reported to decrease amyloid beta production by inhibiting calcium influx, decreasing inflammation and oxidative stress. However, the probable role of this drug in aluminum chloride (AlCl3)-induced experimental dementia is yet to be explored. Therefore, the present study is designed to investigate the effect of AlCl3-induced dementia in mice. Morris water maze test and elevated plus maze were utilized to evaluate learning and memory. Various biochemical estimations including brain acetylcholinesterase activity (AChE), brain total protein, thiobarbituric acid-reactive species (TBARS) level, reduced glutathione (GSH) level, nitrate/nitrite, and superoxide dismutase (SOD) were measured. AlCl3 significantly impaired learning and memory and increased brain AChE,
* Neha [email protected] Anu Rani [email protected] Rupinder K. Sodhi [email protected] Amanpreet Kaur [email protected] 1
Pharmacology Research Lab, Department of Pharmacology, Chandigarh College of Pharmacy, Landran, Mohali, Punjab 140307, India
2
Department of Pharmacology, Chandigarh College of Pharmacy, Landran, Mohali, Punjab 140307, India
brain total protein, TBARS, and nitrate/nitrite and decreased brain GSH or SOD. On the other hand, treatment with diltiazem significantly reversed AlCl3-induced behavioral and biochemical deficits. The present study indicates the beneficial role of diltiazem in AlCl3-induced dementia Keywords Aluminum chloride . Diltiazem . Oxidative stress . Dementia . Morris water maze
Introduction Dementia is one of the common neurodegenerative disorder derived from a Latin word which means impairment of mental powers due to any disease (Kolavora et al. 2012; Tiwari and Soni 2014). Dementia is a devitalizing disorder which progressively declines the cognitive functioning such as memory, speech, language, judgment, orientation, and learning capacity (Mani and Parle 2009). Alzheimer’s disease (AD) is a heterogeneous and the most common form of dementia, affecting millions of men and women worldwide (Bassil and Mollaei 2012). AD is associated with formation of amyloid beta plaques, tau hyperphosphorylation, neurofibrillary tangles, loss of cholinergic activity, unregulated glutamate signaling, increased oxidative stress, increased level of cholesterol, and chronic inflammation (Humpel 2011). It has been hypothesized that metals such as iron, copper, zinc, lead, lithium, silica, fluoride, mercury, and aluminum are involved in amyloid beta (Aβ) aggregation and neurotoxicity (Nunes et al. 2007). Interaction of these transition metal ions with Aβ promotes deposition of amyloid plaques which results in neurotoxic action (White et al. 2011). It has been evidenced that increased amount of aluminum in brain, that might occur via diet, antacid, cosmetics, toothpastes, inhaled fumes, and particles and through drinking water, may results
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in AD (Enas and Khalil 2010). Aluminum can induce neurotoxicity via many mechanisms such as Aβ accumulation, tau hyperphosphorylation, lipid peroxidation, elevated nitric oxide (NO) release, and impaired neuronal exchange of calcium ions (Goma and Mahrous 2013; Stevanoic et al. 2010). Aluminum is not very toxic at normal levels, but its chronic exposure may lead to speech disturbance, dyspraxia, tremors, psychosis, partial paralysis, loss of memory and cognition, and ultimately death (Buraimoh et al. 2012). Aluminum accumulates in the hippocampus in the forms of amyloid beta plaques and neurofibrillary tangles which underlie the pathogenesis of AD (Aly et al. 2011). Accumulation of aluminum results in reactive oxygen species (ROS) formation which depletes normal antioxidant defense mechanism followed by oxidative stress and lipid peroxidation leading to amyloid deposition (Oguz et al. 2012). Aluminum has been reported to affect the brain enzymatic activity and the level of various biomolecules which shows its neurotoxic action (Sushma et al. 2011; Yuan et al. 2012). It has been reported that chronic intragastric exposure to aluminum results in decreased activity of cytochrome c oxidase and various other electron transport chain complexes in rat brain (Mohan et al. 2009). Furthermore, Al can inhibit the activity of various enzymes such as hexokinase, acid and alkaline phosphatases, phosphor-oxidase, and phosphodiesterase by binding to DNA or RNA molecules (Ochmanski and Barabasz 2000). A number of novel therapeutics have been implicated in treatment of AD. A new approach in the treatment of dementia is calcium channel blockers (CCBs), which have been shown to enhance general cognition on the basis of their ability to reverse experimentally induced amnesia and improve learning and memory in young adult animals (Zupan et al. 1996). CCBs protect AD cells from Aβ oligomer production by inhibiting Ca2+ influx (Wareski et al. 2009). It has also been proposed that agents which inhibit Ca2+ elevations also inhibit the production of inflammatory mediators such as NO, tumor necrosis factor (TNF)-α, and interleukin-1 (IL-1) (Hattori et al. 1995; Hottchkiss and Kark 1994). Diltiazem, a benzothiazepine class of CCBs, has been proposed to be effective in traumatic brain injury, short- and long-term retention c y a n i d e - i n d u c e d n e u r o t o x i c i t y, i n f l a m m a t i o n , pentylenetetrazole-provoked amnesia, and lead toxicity (Gibson et al. 2010; Mathangi and Namasivayam 2003; Papazova et al. 2001; Zhanga et al. 2013). It blocks the entry of Ca2+ through L-type calcium channels and thus interferes with calcium signaling which is involved in amyloid toxicity (Quartermain et al. 2001; Tan et al. 2012). Diltiazem attenuates oxidative stress along with increased levels of NO (Anjaneyulu and Chopra 2005). It has also been proved that the drug is well tolerated and adverse effects are seen in less than 5 % of patients (McAuley and Schroeder 2012). Thus, the voltage-gated L-type CCB, diltiazem, that interrupts Ca2+ influx may be proved useful in therapy of AD because of their
neuroprotective, anti-inflammatory, antioxidant, and calcium antagonism effects. However, the study needs further investigation for its use in AD. Donepezil is a reversible and highly selective inhibitor of acetylcholinesterase (AChE) (Shao 2015). The safety and efficacy of donepezil has been reported in the clinical trials for memory deficits associated with AD and other conditions (Winblad 2009). In this study, donepezil is used as a positive control.
Materials and methods Swiss albino mice (20–30 g) of either sex were employed in the present study (procured from the Indian Institute of Integrative Medicine, Jammu, India) and were housed in departmental animal house. Mice were maintained at standard laboratory pellet chow diet and water ad libitum. The mice were exposed to 12-h light and 12-h dark cycle. The animals were acclimatized to laboratory conditions prior to the experimental study. The experiment was performed between 0930 and 1730 hours in semi-soundproof laboratory conditions. All the experiments were performed in accordance with the guidelines of the Institutional Animal Ethical Committee (IAEC). Adequate measures were taken to minimize pain or discomfort with animal experimental procedures. The care of animals was carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India (Reg. No. 1201/9/08/CPCSEA).
Drugs and reagents All the drugs and reagents used in the study were freshly prepared. Aluminum chloride (AlCl3) was purchased from Loba Chemicals, Mumbai, India. Diltiazem was purchased from TCI chemicals Pvt. Ltd., Chennai, India. 5,5-Dithiobis2-nitrobenzoic acid (DTNB), Folin-Ciocalteu phenol reagent, bovine serum albumin, pyridine, reduced glutathione (GSH), sulfanilamide, n-napthyl ethylenediamide dichloride, and dipotassium hydrogen phosphate were purchased from Loba Chemicals, Mumbai, India. Donepezil and thiobarbituric acid were purchased from Magus Chemicals, Mohali, India. Trichloroacetic acid was purchased from Nice Chem. Pvt. Ltd., Cochin, India. EDTA was purchased from Thomas Baker, India. Diltiazem was dissolved in distilled water and given by oral route. AlCl3 was dissolved in 0.9 % normal saline solution and was also given by oral route. Donepezil was dissolved in saline and was administered by intraperitoneal route (i.p.).
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Induction of experimental dementia by AlCl3 AlCl3 was chronically administered to animals at a dose of 300 mg/kg/day by oral route (p.o.) for 1 month to induce dementia (Enas and Khalil 2010).
Laboratory models Exteroceptive behavioral models Exteroceptive behavioral models are as follows: & &
Morris water maze test Elevated plus maze test
Morris water maze test Morris water maze test was employed to access the learning and memory of mice (Morris 1984; Saraf et al. 2011). Morris water maze is a swimming-based model where the animals learn to escape on to a hidden platform. It consists of a large circular pool (150 cm in diameter and 45 cm in height), filled to a depth of 30 cm with water at 28 ± 1 °C. The water was made opaque by using white non-toxic dye or milk. The tank was divided in four quadrants with the help of two threads. The water pool was placed in illuminated light room. A submerged platform (10 cm2) painted white was fixed at a right angle to each other on the rim of the pool placed inside the target quadrant of this pool 1 cm below surface of water. The position of the platform was kept unaltered throughout the training session. Each animal was subjected to four consecutive trials with the inter-trial gap of 5 min. The mice were gently placed in the water between quadrants, facing the wall of the pool with drop location changing for each trial, and allowed 120 s to locate the platform. Then, it was allowed to stay on the platform for 20 s. When an animal failed to find the platform within 120 s, the experimenter guided the same to reach onto the platform and allowed to remain there for 20 s. The escape latency time (ELT) to locate the hidden platform in the water maze on day 4 was noted as an index of acquisition or learning. On the 5th day, the platform was removed and the time spent by an animal in each quadrant was noted. The time spent by the animal in target quadrant (Q4) in search of missing platform was noted as an index of retrieval or memory.
arms are connected with a central square of 5 × 5 cm. The entire maze is placed 25-cm high above the ground. Each mouse was placed individually at one end of the open arm facing away from the central square. The time taken by the animal to move from the open arm to the closed arm was recorded as initial transfer latency (ITL) time. The animal was allowed to explore the maze for 10 s after recording ITL. If the animal did not enter the enclosed arm within 90 s, the same was guided to the enclosed arm and ITL was recorded as 90 s. Biochemical estimations Animals were sacrificed by cervical dislocation; brains were removed and homogenized in phosphate buffer (pH = 7.4). The homogenates were than centrifuged at 3000 rpm for 15 min. The supernatant of homogenates was used for biochemical estimations. Estimation of brain AChE activity The whole brain AChE activity was measured spectrophotometrically (UV-1800 spectrophotometer, Shimadzu, Japan) at 420 nm by the method of Ellman et al. (1961) with slight modifications (Sain et al. 2011). Estimation of brain total protein content The brain total protein was determined by the method of Lowry et al. (1951). Estimation of thiobarbituric acid-reactive species level The quantitative measurement of thiobarbituric acid-reactive species (TBARS), an index of lipid peroxidation in the brain, was performed according to the method of Ohokawa et al. (1979). Estimation of GSH The reduced GSH content in the brain was estimated spectrophotometrically (UV-1800 spectrophotometer, Shimadzu, Japan) at 412 nm using the method of Beutler et al. (1963).
Elevated plus maze test Estimation of nitrite concentration Elevated plus maze was used to study the locomotory activity of the mice (Kumar et al. 2011; Parle and Singh 2007). It consists of two opposite white open arms (16 × 5 cm), crossed with two closed walls (16 × 5 cm) with 12-cm-high walls. The
Brain nitrite/nitrate concentration was measured spectrophotometrically (UV-1800 spectrophotometer, Shimadzu, Japan) at 542 nm, using the method of Sastry et al. (2002).
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Estimation of superoxide dismutase level Superoxide dismutase (SOD) activity was determined by the method of Wang et al. (1998).
Experimental protocol Eight groups of mice were employed in the study. Each group comprised minimum 8 mice, n = 8. Group I (control, n = 8) Mice were administered with distilled water (10 ml/kg, p.o.) 30 min before acquisition trials, conducted from day 1 to day 4, and 30 min before retrieval trial conducted on day 5 using the Morris water maze (MWM) test.
Group VII (AlCl3 + diltiazem high dose (40 mg/kg, p.o.), n = 8) AlCl3-treated mice were administered with diltiazem (40 mg/kg, p.o.) for 14 days and then subjected to MWM test. The rest of the procedure was the same as described in group VI. Group VIII (AlCl3 + donepezil, n = 8) AlCl3-treated mice were administered donepezil (0.1 mg/kg/day, i.p.) for 14 days and then subjected to MWM test. The coadministration of donepezil along with AlCl3 was continued from day 1 to day 4 during acquisition trials. Animals were administered with distilled water (10 ml/kg; p.o.) 30 min before retrieval trial, i.e., on day 5. Statistical analysis
Group II (normal saline, n = 8) Mice were administered 0.9 % w/v normal saline (10 ml/kg, p.o.) daily for 14 days and then subjected to MWM test. The vehicle was also administered (30 min) before acquisition trials, conducted from day 1 to day 4, and before retrieval trial, conducted on day 5, (day 19).
The results were expressed as mean ± standard error of means (SEM). The data obtained from various groups were statistically analyzed using one-way ANOVA followed by Tukey’s multiple range test. p < 0.05 was considered to be statistically significant.
Group III (donepezil per se, n = 8) Mice were administered with donepezil (0.1 mg/kg/day i.p.) for 14 days and then subjected to MWM test. Donepezil was administered during acquisition trial, conducted from day 1 to day 4. The animals were administered distilled water (10 ml/kg, p.o.) 30 min before retrieval trial, conducted on day 5 (day 19).
Results
Group IV (diltiazem per se, n = 8) Mice were administered diltiazem (20 mg/kg/day p.o.) for 14 days and then subjected to MWM test. The treatment was continued during acquisition trial, conducted from day 1 to day 4 (day 15 to day 18). The animals were administered 0.9 % w/v normal saline (10 ml/kg, p.o.) 30 min before retrieval trial, conducted on day 5. Group V (AlCl3 control, n = 8) Mice were administered with AlCl3 (300 mg/kg/day, p.o.) for 1 month followed by exposure to MWM test. AlCl3 was also administered during acquisition trial, conducted from day 1 to day 4. The animals were administered 0.9 % w/v normal saline (10 ml/kg; p.o.) 30 min before retrieval trial, conducted on day 5. Group VI (AlCl3 + diltiazem low dose (20 mg/kg, p.o.), n = 8) AlCl3-treated mice were administered with diltiazem (20 mg/kg, p.o.) for 14 days and then subjected to MWM test. The coadministration of diltiazem along with AlCl3 was continued from day 1 to day 4 during acquisition trials. Animals were administered with 0.9 % w/v normal saline (10 ml/kg; p.o.) 30 min before retrieval trial, i.e., on day 5.
Effect on body weight Mice treated with AlCl3 (300 mg/kg; p.o.) for 1 month showed a significant decrease in body weight as compared to the control animals fed on normal diet (Fig. 1). A significant increase in body weight was observed after administration of diltiazem (20 and 40 mg/kg; p.o.) and donepezil (0.1 mg/kg; i.p.) in comparison to AlCl3-treated animals (Fig. 1). Effect on transfer latency time using elevated plus maze Normal control mice showed a significant decrease in day 2 transfer latency time (TLT) as compared to day 1 TLT (Fig. 2). Mice treated with AlCl3 (300 mg/kg; p.o.) for 1 month showed a significant increase in day 2 TLT in comparison to normal animals (Fig. 2). A significant fall in day 2 TLT was observed after administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg; i.p.) in AlCl3-treated mice (Fig. 2). Effect of vehicle on ELT and mean time spent in target quadrant using MWM test Control mice showed a significant decline in their day 4 ELT on subsequent exposure to MWM when compared to day 1 ELT reflecting normal acquisition (Fig. 3). Moreover, these animals showed a significant increase in time spent in target quadrant (TSTQ) in comparison to time spent in other
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Fig. 1 Effect of diltiazem on body weight of AlCl3-treated mice. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus body weight of control; b p < 0.05 versus body weight of aluminum chloride-treated group
quadrants conducted on day 5, reflecting normal retrieval (Fig. 4). Vehicle, i.e., normal saline 0.9 % w/v (10 ml/kg), did not show any significant effect on day 4 ELT (Fig. 3) and day 5 TSTQ (Fig. 4) when compared to control animals. Mice treated with AlCl3 (300 mg/kg; p.o.) for 1 month showed a significant increase in day 4 ELT values (Fig. 3) and decrease in day 5 TSTQ (Fig. 4) in comparison to normal control animals indicating impairment in learning and memory. Administration of diltiazem (20 and 40 mg/kg; p.o.)/ donepezil (0.1 mg/kg; i.p.) in AlCl3-treated mice exhibited a
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Fig. 2 Effect of diltiazem on TLT of AlCl3-treated mice using elevated plus maze. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl 3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus day 1 TLT in control group; bp < 0.05 versus day 2 TLT in control group; c p < 0.05 versus day 2 TLT in AlCl3-treated group
DAY 4 ELT (sec)
Fig. 3 Effect of diltiazem on day 4 ELT of AlCl3-treated mice using Morris water maze. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.), D.H2O distilled water. Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus day 1 ELT in control group; bp < 0.05 versus day 4 ELT in control group; cp < 0.05 versus day 4 ELT in AlCl3-treated group
significant fall in day 4 ELT (Fig. 3) and rise in day 5 TSTQ (Fig. 4) indicating reversal of learning and memory. Furthermore, diltiazem per se and donepezil per se did not show any significant effect on day 4 ELT (Fig. 3) and day 5 TSTQ (Fig. 4). Effect on brain AChE activity AlCl3-treated mice showed a significant rise in brain AChE activity when compared to the control group (Fig. 5). Administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg; i.p.) prevented AlCl3-induced increase in brain AChE activity (Fig. 5). However, diltiazem per se, donepezil per se, and vehicle groups did not show any effect on brain AChE activity (Fig. 5). Effect on brain TBARS level AlCl3-treated mice showed a significant increase in brain TBARS level when compared to the control group (Fig. 6). Administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg; i.p.) prevented AlCl3-induced increase in brain TBARS level (Fig. 6). Furthermore, diltiazem per se, donepezil per se, and vehicle groups did not show any effect on brain TBARS level (Fig. 6). Effect on brain reduced GSH level AlCl3-treated mice showed a significant decline in brain reduced GSH level in comparison with control group animals (Fig. 7). Administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg, i.p.) prevented
Fig. 4 Effect of diltiazem on mean time spent in target quadrant (TSTQ) (Q4) of AlCl3-treated mice using Morris water maze (MWM) test. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.),
Mean Time Spent in Target Quadrant (sec)
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AlCl3-induced decrease in brain GSH level (Fig. 7). However, administration of diltiazem per se, donepezil per se, and normal saline did not show any significant changes in brain reduced GSH level when compared with control group animals (Fig. 7).
Effect on brain nitrite/nitrate level AlCl3-treated mice showed a significant rise in brain nitrite/ nitrate level when compared to the control group (Fig. 8). Administration of diltiazem (20 and 40 mg/kg; p.o.)/ donepezil (0.1 mg/kg; i.p.) prevented AlCl3-induced increase in brain nitrite/nitrate level (Fig. 8). Diltiazem per se, donepezil per se, and normal saline group did not show any significant effect on brain nitrite/nitrate level when compared to control group (Fig. 8). 200 AChE activity (nM/min/mg of protein)
Fig. 5 Effect of diltiazem on brain acetylcholinesterase (AChE) activity in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± Standard error of mean (SEM), n = 8, oneway ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus brain AChE activity in control, bp < 0.05 versus brain AChE activity in AlCl3-treated group
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Effect on brain superoxide dismutase activity AlCl3-treated mice showed a significant decrease in brain superoxide dismutase (SOD) activity in comparison to control group animals (Fig. 9). Administration of diltiazem (20 and 40 mg/kg, p.o.)/donepezil (0.1 mg/kg, i.p.) to AlCl3-treated mice showed a significant rise in brain superoxide dismutase activity (Fig. 9). However, administration of diltiazem per se, donepezil per se, and normal saline did not show any significant variation in brain superoxide dismutase activity when compared with normal control animals (Fig. 9).
Discussion In present study, MWM and elevated plus maze are used to evaluate learning and memory in experimental animals. In a b
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Brain TBARS (nmol/mg of protein)
Fig. 6 Effect of diltiazem on brain thiobarbituric acid-reactive species (TBARS) level in AlCl3treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus brain TBARS level in control; bp < 0.05 versus brain TBARS level in AlCl3-treated group
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MWM, fall in day 4 ELT and rise in day 5 TSTQ in search of platform indicate memory retrieval. In elevated plus maze, fall in day 2 TLT indicates normal learning and memory. The normal control mice showed decreased day 4 ELT and increased day 5 TSTQ using MWM indicating normal learning and memory. Further, control mice show decreased day 2 TLT in elevated plus maze indicating no impairment in memory. Administration of vehicle, i.e., normal saline, did not show any additional effect on memory retrieval. Furthermore, no per se effects are seen on acquisition and retrieval in comparison to the normal group. These results agree with the results of previous studies done in other laboratories (Kumar et al. 2011; Saraf et al. 2011). AlCl3 (300 mg/kg; p.o.) was administered for 30 days and resulted in memory impairment in mice as indicated by a significant increase in day 4 ELT and decrease in day 5 30 Brain GSH (micromoles/mg of protein)
Fig. 7 Effect of diltiazem on brain reduced glutathione (GSH) level in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, oneway ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus brain GSH level in control; bp < 0.05 versus brain GSH level in AlCl3-treated group
TSTQ. AlCl3 administration also increased day 2 TLT which also indicates impaired learning and memory. Moreover, AlCl3-treated animals showed a significant increase in brain AChE activity, brain nitrite/nitrate levels, and brain TBARS levels along with depletion of brain SOD and GSH levels. In the present study, a marked decrease in body weight in aluminum exposed animals was observed as compared to the animals of control group. These findings are also in line with previous studies done in other laboratories (Cherrot et al. 1995; Sharma et al. 2007). Aluminum is a ubiquitous metal that has been implicated in the etiology of neurodegenerative disorders where it exacerbates brain oxidative damage, neuroinflammation, and Aβ deposition (Becaria et al. 2002). Aluminum exposure exerted adverse effects on learning and memory which were manifested by increase in number of acquisition and
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Fig. 8 Effect of diltiazem on brain nitrite/nitrate level in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus brain nitrite/nitrate level in control; b p < 0.05 versus brain nitrite/ nitrate in AlCl3-treated group
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retention errors of rodents. Aluminum causes disturbances in cholinergic neurotransmission and disrupts the cognitive behavior of animals by increasing the stress levels. The elevations in AChE are either direct result of neurotoxic effect of metals or due to increased lipid peroxidation (Kaizer et al. 2008). Studies have shown that aluminum exposure results in decrease of hexokinase activity, which in turn decreases the pyruvate formation and hence affects the synthesis of acetylcholine and subsequently the enzyme activity of AChE. Aluminum is a pro-oxidant and indirectly results in the production of free radicals leading to oxidative damage and reduced levels of ROS which indirectly affect acetylcholinesterase enzyme activity (Yuan et al. 2012). Aluminum causes marked oxidative damage by increasing the redox active iron concentration in the brain mainly via the Fenton reaction (Exley 2004). Aluminum causes reduced axonal mitochondria
turnover, disruption of Golgi, and reduction of synaptic vesicles which result in release of oxidative products like malondialdehyde, carbonyls, peroxynitrites, and enzymes like SOD within the neurons (Yuan et al. 2012). Aluminum can also induce neurotoxicity via impairing neuronal exchange of calcium ions and increase Ca2+ level in brain (Goma and Mahrous 2013). The increased Ca2+ causes mitochondrial overload, excessive production of Aβ, and tau hyperphosphorylation which further generate caspases and cytochrome c and increase ROS production leading to cell death and neurodegeneration (Stutzmann 2007). Administration of diltiazem (20 and 40 mg/kg p.o. for 14 days) to AlCl3-treated mice results in decreased day 4 ELT and increased day 5 TSTQ which indicate improved learning and memory. Diltiazem administration also showed a significant decrease in day 2 TLT. Further, diltiazem reduces
60 SOD activity (picomole/min/mg og wet tissue)
Fig. 9 Effect of diltiazem on brain superoxide dismutase (SOD) activity in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, oneway ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus brain SOD activity in control; bp < 0.05 versus brain SOD activity in AlCl3-treated group
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brain AChE, brain TBARS, and brain nitrite/nitrate levels and increases brain GSH and SOD activity. Diltiazem administration also improved the body weight in AlCl3-treated mice. Moreover, administration of donepezil (0.1 mg/kg; i.p. for 14 days) attenuates the brain changes induced by administration of AlCl3. These results are consistent with the results of previous studies (Packard et al. 1996). In the present study, donepezil has been used as a positive control. Diltiazem, a CCB drug, has been evidenced to be useful in hypertension, angina, and cardiovascular disease. Diltiazem has vasodilatory effects on arterial vessels and it increases the reflex in sympathetic response. Its overall effect in CVD is a drop in blood pressure and an increase in cardiac output, heart rate and contractility (Gura 2008). Other than CVD, diltiazem has also been suggested to be useful in general cognitive enhancement on the basis of its ability to improve learning and memory in adult animals (Quartermain et al. 2001). The concentrations (dose) employed in the study were chosen after performing the pilot studies starting with very low dose and referring to the previous reports. A range of concentrations have been used in animals varying from as low as 5 mg/kg to as high as 1000 mg/kg for 40–60 days (Biala and Kruk 2009; Bishnoi et al. 2008; Correa et al. 2009). Voltage-gated calcium channels play an important role in the entry of Ca2+ ions into excitable cells and are also involved in a series of calcium-dependent processes, such as muscle contraction, hormone or neurotransmitter release, gene expression, and cell death (Augustine et al. 1987; Miller 1987). Voltage-gated calcium channels are blocked by 1,4dihydropyridines (DHPs) such as nifedipine, phenylalkylamines such as verapamil, and benzothiazepines such as diltiazem (Shaldam et al. 2014). They are multisubunit complexes, comprising alpha-1, alpha-2, beta, and delta subunits. The channel activity is directed by the pore-forming and voltage-sensitive alpha-1 subunit. In many cases, this subunit is sufficient to generate voltage-sensitive calcium channel activity that displays the major electrophysiological and pharmacological properties of the full-fledged heteromeric channels (Soldatov et al. 1995; Zuhlke et al. 1998). It has been proposed to be useful in Alzheimer’s disease because of its calcium-lowering effect across the voltage-gated channels. Increased calcium levels are postulated in increased mitochondrial overload, oxidative damage, Aβ production, and tau phosphorylation (Iqbal and Grundke 2007). Increased Aβ further interacts with Fe2+ and Cu2+ to generate ROS, and ROS leads to lipid peroxidation (Zhu et al. 2006). Diltiazem thus, by blocking Ca2+ effects, avoids these adverse effects of calcium dysregulation. Furthermore, diltiazem also showed antiinflammatory effects by stimulating production of antiinflammatory mediators IL-10 and inhibiting proinflammatory mediator IL-6 (Dubey and Hesong 2006). Even though, numerous studies have suggested the neuroprotective potential of calcium channel blockers in disorders such
as parkinsonism (Ritz et al. 2010), Alzheimer’s disease (Anekonda and Quinn 2011), focal cerebral ischemia (Takahara et al. 2004) and spinal cord ischemia-reperfusion injury (Fansa et al. 2009). However, till date, none of the reports suggest the memory restorative ability of diltiazem. This is the first report in itself that speculates the protective role of diltiazem in memory and learning, although this also necessitates in-depth evaluation of the exact mechanism of diltiazem in dementia. Thus, from the literature survey and data in hand, it may be concluded that diltiazem has a neuroprotective effect on AlCl3-induced behavioral and biochemical parameters in mice.
Conclusion The results obtained from our study indicate that diltiazem significantly improves AlCl3-induced memory impairment and biochemical changes. This is the first report indicating the memory-restorative ability of a calcium channel blocker in metal-induced dementia. The improvement in learning and memory produced may be owed to its anti-oxidative and anticholinesterase potential. However in-depth in vitro and in vivo studies need to be performed to evaluate the exact mechanism of diltiazem in memory enhancement.
Acknowledgments The authors are thankful to Chandigarh College of Pharmacy, Landran (India) for supporting this study and providing technical facilities for the work.
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ORIGINAL ARTICLE
Protective effect of a calcium channel blocker “diltiazem” on aluminum chloride-induced dementia in mice Anu Rani 1 & Neha 2
&
Rupinder K. Sodhi 2 & Amanpreet Kaur 1
Received: 11 May 2015 / Accepted: 19 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Many studies report that heavy metals such as aluminum are involved in amyloid beta aggregation and neurotoxicity. Further, high concentration of aluminum in the brain deregulates calcium signaling which contributes to synaptic dysfunction and halts neuronal communication which ultimately leads to the development of Alzheimer’s disease. Recently, diltiazem, a calcium channel blocker clinically used in angina, is reported to decrease amyloid beta production by inhibiting calcium influx, decreasing inflammation and oxidative stress. However, the probable role of this drug in aluminum chloride (AlCl3)-induced experimental dementia is yet to be explored. Therefore, the present study is designed to investigate the effect of AlCl3-induced dementia in mice. Morris water maze test and elevated plus maze were utilized to evaluate learning and memory. Various biochemical estimations including brain acetylcholinesterase activity (AChE), brain total protein, thiobarbituric acid-reactive species (TBARS) level, reduced glutathione (GSH) level, nitrate/nitrite, and superoxide dismutase (SOD) were measured. AlCl3 significantly impaired learning and memory and increased brain AChE,
* Neha [email protected] Anu Rani [email protected] Rupinder K. Sodhi [email protected] Amanpreet Kaur [email protected] 1
Pharmacology Research Lab, Department of Pharmacology, Chandigarh College of Pharmacy, Landran, Mohali, Punjab 140307, India
2
Department of Pharmacology, Chandigarh College of Pharmacy, Landran, Mohali, Punjab 140307, India
brain total protein, TBARS, and nitrate/nitrite and decreased brain GSH or SOD. On the other hand, treatment with diltiazem significantly reversed AlCl3-induced behavioral and biochemical deficits. The present study indicates the beneficial role of diltiazem in AlCl3-induced dementia Keywords Aluminum chloride . Diltiazem . Oxidative stress . Dementia . Morris water maze
Introduction Dementia is one of the common neurodegenerative disorder derived from a Latin word which means impairment of mental powers due to any disease (Kolavora et al. 2012; Tiwari and Soni 2014). Dementia is a devitalizing disorder which progressively declines the cognitive functioning such as memory, speech, language, judgment, orientation, and learning capacity (Mani and Parle 2009). Alzheimer’s disease (AD) is a heterogeneous and the most common form of dementia, affecting millions of men and women worldwide (Bassil and Mollaei 2012). AD is associated with formation of amyloid beta plaques, tau hyperphosphorylation, neurofibrillary tangles, loss of cholinergic activity, unregulated glutamate signaling, increased oxidative stress, increased level of cholesterol, and chronic inflammation (Humpel 2011). It has been hypothesized that metals such as iron, copper, zinc, lead, lithium, silica, fluoride, mercury, and aluminum are involved in amyloid beta (Aβ) aggregation and neurotoxicity (Nunes et al. 2007). Interaction of these transition metal ions with Aβ promotes deposition of amyloid plaques which results in neurotoxic action (White et al. 2011). It has been evidenced that increased amount of aluminum in brain, that might occur via diet, antacid, cosmetics, toothpastes, inhaled fumes, and particles and through drinking water, may results
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in AD (Enas and Khalil 2010). Aluminum can induce neurotoxicity via many mechanisms such as Aβ accumulation, tau hyperphosphorylation, lipid peroxidation, elevated nitric oxide (NO) release, and impaired neuronal exchange of calcium ions (Goma and Mahrous 2013; Stevanoic et al. 2010). Aluminum is not very toxic at normal levels, but its chronic exposure may lead to speech disturbance, dyspraxia, tremors, psychosis, partial paralysis, loss of memory and cognition, and ultimately death (Buraimoh et al. 2012). Aluminum accumulates in the hippocampus in the forms of amyloid beta plaques and neurofibrillary tangles which underlie the pathogenesis of AD (Aly et al. 2011). Accumulation of aluminum results in reactive oxygen species (ROS) formation which depletes normal antioxidant defense mechanism followed by oxidative stress and lipid peroxidation leading to amyloid deposition (Oguz et al. 2012). Aluminum has been reported to affect the brain enzymatic activity and the level of various biomolecules which shows its neurotoxic action (Sushma et al. 2011; Yuan et al. 2012). It has been reported that chronic intragastric exposure to aluminum results in decreased activity of cytochrome c oxidase and various other electron transport chain complexes in rat brain (Mohan et al. 2009). Furthermore, Al can inhibit the activity of various enzymes such as hexokinase, acid and alkaline phosphatases, phosphor-oxidase, and phosphodiesterase by binding to DNA or RNA molecules (Ochmanski and Barabasz 2000). A number of novel therapeutics have been implicated in treatment of AD. A new approach in the treatment of dementia is calcium channel blockers (CCBs), which have been shown to enhance general cognition on the basis of their ability to reverse experimentally induced amnesia and improve learning and memory in young adult animals (Zupan et al. 1996). CCBs protect AD cells from Aβ oligomer production by inhibiting Ca2+ influx (Wareski et al. 2009). It has also been proposed that agents which inhibit Ca2+ elevations also inhibit the production of inflammatory mediators such as NO, tumor necrosis factor (TNF)-α, and interleukin-1 (IL-1) (Hattori et al. 1995; Hottchkiss and Kark 1994). Diltiazem, a benzothiazepine class of CCBs, has been proposed to be effective in traumatic brain injury, short- and long-term retention c y a n i d e - i n d u c e d n e u r o t o x i c i t y, i n f l a m m a t i o n , pentylenetetrazole-provoked amnesia, and lead toxicity (Gibson et al. 2010; Mathangi and Namasivayam 2003; Papazova et al. 2001; Zhanga et al. 2013). It blocks the entry of Ca2+ through L-type calcium channels and thus interferes with calcium signaling which is involved in amyloid toxicity (Quartermain et al. 2001; Tan et al. 2012). Diltiazem attenuates oxidative stress along with increased levels of NO (Anjaneyulu and Chopra 2005). It has also been proved that the drug is well tolerated and adverse effects are seen in less than 5 % of patients (McAuley and Schroeder 2012). Thus, the voltage-gated L-type CCB, diltiazem, that interrupts Ca2+ influx may be proved useful in therapy of AD because of their
neuroprotective, anti-inflammatory, antioxidant, and calcium antagonism effects. However, the study needs further investigation for its use in AD. Donepezil is a reversible and highly selective inhibitor of acetylcholinesterase (AChE) (Shao 2015). The safety and efficacy of donepezil has been reported in the clinical trials for memory deficits associated with AD and other conditions (Winblad 2009). In this study, donepezil is used as a positive control.
Materials and methods Swiss albino mice (20–30 g) of either sex were employed in the present study (procured from the Indian Institute of Integrative Medicine, Jammu, India) and were housed in departmental animal house. Mice were maintained at standard laboratory pellet chow diet and water ad libitum. The mice were exposed to 12-h light and 12-h dark cycle. The animals were acclimatized to laboratory conditions prior to the experimental study. The experiment was performed between 0930 and 1730 hours in semi-soundproof laboratory conditions. All the experiments were performed in accordance with the guidelines of the Institutional Animal Ethical Committee (IAEC). Adequate measures were taken to minimize pain or discomfort with animal experimental procedures. The care of animals was carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India (Reg. No. 1201/9/08/CPCSEA).
Drugs and reagents All the drugs and reagents used in the study were freshly prepared. Aluminum chloride (AlCl3) was purchased from Loba Chemicals, Mumbai, India. Diltiazem was purchased from TCI chemicals Pvt. Ltd., Chennai, India. 5,5-Dithiobis2-nitrobenzoic acid (DTNB), Folin-Ciocalteu phenol reagent, bovine serum albumin, pyridine, reduced glutathione (GSH), sulfanilamide, n-napthyl ethylenediamide dichloride, and dipotassium hydrogen phosphate were purchased from Loba Chemicals, Mumbai, India. Donepezil and thiobarbituric acid were purchased from Magus Chemicals, Mohali, India. Trichloroacetic acid was purchased from Nice Chem. Pvt. Ltd., Cochin, India. EDTA was purchased from Thomas Baker, India. Diltiazem was dissolved in distilled water and given by oral route. AlCl3 was dissolved in 0.9 % normal saline solution and was also given by oral route. Donepezil was dissolved in saline and was administered by intraperitoneal route (i.p.).
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Induction of experimental dementia by AlCl3 AlCl3 was chronically administered to animals at a dose of 300 mg/kg/day by oral route (p.o.) for 1 month to induce dementia (Enas and Khalil 2010).
Laboratory models Exteroceptive behavioral models Exteroceptive behavioral models are as follows: & &
Morris water maze test Elevated plus maze test
Morris water maze test Morris water maze test was employed to access the learning and memory of mice (Morris 1984; Saraf et al. 2011). Morris water maze is a swimming-based model where the animals learn to escape on to a hidden platform. It consists of a large circular pool (150 cm in diameter and 45 cm in height), filled to a depth of 30 cm with water at 28 ± 1 °C. The water was made opaque by using white non-toxic dye or milk. The tank was divided in four quadrants with the help of two threads. The water pool was placed in illuminated light room. A submerged platform (10 cm2) painted white was fixed at a right angle to each other on the rim of the pool placed inside the target quadrant of this pool 1 cm below surface of water. The position of the platform was kept unaltered throughout the training session. Each animal was subjected to four consecutive trials with the inter-trial gap of 5 min. The mice were gently placed in the water between quadrants, facing the wall of the pool with drop location changing for each trial, and allowed 120 s to locate the platform. Then, it was allowed to stay on the platform for 20 s. When an animal failed to find the platform within 120 s, the experimenter guided the same to reach onto the platform and allowed to remain there for 20 s. The escape latency time (ELT) to locate the hidden platform in the water maze on day 4 was noted as an index of acquisition or learning. On the 5th day, the platform was removed and the time spent by an animal in each quadrant was noted. The time spent by the animal in target quadrant (Q4) in search of missing platform was noted as an index of retrieval or memory.
arms are connected with a central square of 5 × 5 cm. The entire maze is placed 25-cm high above the ground. Each mouse was placed individually at one end of the open arm facing away from the central square. The time taken by the animal to move from the open arm to the closed arm was recorded as initial transfer latency (ITL) time. The animal was allowed to explore the maze for 10 s after recording ITL. If the animal did not enter the enclosed arm within 90 s, the same was guided to the enclosed arm and ITL was recorded as 90 s. Biochemical estimations Animals were sacrificed by cervical dislocation; brains were removed and homogenized in phosphate buffer (pH = 7.4). The homogenates were than centrifuged at 3000 rpm for 15 min. The supernatant of homogenates was used for biochemical estimations. Estimation of brain AChE activity The whole brain AChE activity was measured spectrophotometrically (UV-1800 spectrophotometer, Shimadzu, Japan) at 420 nm by the method of Ellman et al. (1961) with slight modifications (Sain et al. 2011). Estimation of brain total protein content The brain total protein was determined by the method of Lowry et al. (1951). Estimation of thiobarbituric acid-reactive species level The quantitative measurement of thiobarbituric acid-reactive species (TBARS), an index of lipid peroxidation in the brain, was performed according to the method of Ohokawa et al. (1979). Estimation of GSH The reduced GSH content in the brain was estimated spectrophotometrically (UV-1800 spectrophotometer, Shimadzu, Japan) at 412 nm using the method of Beutler et al. (1963).
Elevated plus maze test Estimation of nitrite concentration Elevated plus maze was used to study the locomotory activity of the mice (Kumar et al. 2011; Parle and Singh 2007). It consists of two opposite white open arms (16 × 5 cm), crossed with two closed walls (16 × 5 cm) with 12-cm-high walls. The
Brain nitrite/nitrate concentration was measured spectrophotometrically (UV-1800 spectrophotometer, Shimadzu, Japan) at 542 nm, using the method of Sastry et al. (2002).
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Estimation of superoxide dismutase level Superoxide dismutase (SOD) activity was determined by the method of Wang et al. (1998).
Experimental protocol Eight groups of mice were employed in the study. Each group comprised minimum 8 mice, n = 8. Group I (control, n = 8) Mice were administered with distilled water (10 ml/kg, p.o.) 30 min before acquisition trials, conducted from day 1 to day 4, and 30 min before retrieval trial conducted on day 5 using the Morris water maze (MWM) test.
Group VII (AlCl3 + diltiazem high dose (40 mg/kg, p.o.), n = 8) AlCl3-treated mice were administered with diltiazem (40 mg/kg, p.o.) for 14 days and then subjected to MWM test. The rest of the procedure was the same as described in group VI. Group VIII (AlCl3 + donepezil, n = 8) AlCl3-treated mice were administered donepezil (0.1 mg/kg/day, i.p.) for 14 days and then subjected to MWM test. The coadministration of donepezil along with AlCl3 was continued from day 1 to day 4 during acquisition trials. Animals were administered with distilled water (10 ml/kg; p.o.) 30 min before retrieval trial, i.e., on day 5. Statistical analysis
Group II (normal saline, n = 8) Mice were administered 0.9 % w/v normal saline (10 ml/kg, p.o.) daily for 14 days and then subjected to MWM test. The vehicle was also administered (30 min) before acquisition trials, conducted from day 1 to day 4, and before retrieval trial, conducted on day 5, (day 19).
The results were expressed as mean ± standard error of means (SEM). The data obtained from various groups were statistically analyzed using one-way ANOVA followed by Tukey’s multiple range test. p < 0.05 was considered to be statistically significant.
Group III (donepezil per se, n = 8) Mice were administered with donepezil (0.1 mg/kg/day i.p.) for 14 days and then subjected to MWM test. Donepezil was administered during acquisition trial, conducted from day 1 to day 4. The animals were administered distilled water (10 ml/kg, p.o.) 30 min before retrieval trial, conducted on day 5 (day 19).
Results
Group IV (diltiazem per se, n = 8) Mice were administered diltiazem (20 mg/kg/day p.o.) for 14 days and then subjected to MWM test. The treatment was continued during acquisition trial, conducted from day 1 to day 4 (day 15 to day 18). The animals were administered 0.9 % w/v normal saline (10 ml/kg, p.o.) 30 min before retrieval trial, conducted on day 5. Group V (AlCl3 control, n = 8) Mice were administered with AlCl3 (300 mg/kg/day, p.o.) for 1 month followed by exposure to MWM test. AlCl3 was also administered during acquisition trial, conducted from day 1 to day 4. The animals were administered 0.9 % w/v normal saline (10 ml/kg; p.o.) 30 min before retrieval trial, conducted on day 5. Group VI (AlCl3 + diltiazem low dose (20 mg/kg, p.o.), n = 8) AlCl3-treated mice were administered with diltiazem (20 mg/kg, p.o.) for 14 days and then subjected to MWM test. The coadministration of diltiazem along with AlCl3 was continued from day 1 to day 4 during acquisition trials. Animals were administered with 0.9 % w/v normal saline (10 ml/kg; p.o.) 30 min before retrieval trial, i.e., on day 5.
Effect on body weight Mice treated with AlCl3 (300 mg/kg; p.o.) for 1 month showed a significant decrease in body weight as compared to the control animals fed on normal diet (Fig. 1). A significant increase in body weight was observed after administration of diltiazem (20 and 40 mg/kg; p.o.) and donepezil (0.1 mg/kg; i.p.) in comparison to AlCl3-treated animals (Fig. 1). Effect on transfer latency time using elevated plus maze Normal control mice showed a significant decrease in day 2 transfer latency time (TLT) as compared to day 1 TLT (Fig. 2). Mice treated with AlCl3 (300 mg/kg; p.o.) for 1 month showed a significant increase in day 2 TLT in comparison to normal animals (Fig. 2). A significant fall in day 2 TLT was observed after administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg; i.p.) in AlCl3-treated mice (Fig. 2). Effect of vehicle on ELT and mean time spent in target quadrant using MWM test Control mice showed a significant decline in their day 4 ELT on subsequent exposure to MWM when compared to day 1 ELT reflecting normal acquisition (Fig. 3). Moreover, these animals showed a significant increase in time spent in target quadrant (TSTQ) in comparison to time spent in other
Naunyn-Schmiedeberg's Arch Pharmacol 30 25 Body Weight (g)
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Fig. 1 Effect of diltiazem on body weight of AlCl3-treated mice. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus body weight of control; b p < 0.05 versus body weight of aluminum chloride-treated group
quadrants conducted on day 5, reflecting normal retrieval (Fig. 4). Vehicle, i.e., normal saline 0.9 % w/v (10 ml/kg), did not show any significant effect on day 4 ELT (Fig. 3) and day 5 TSTQ (Fig. 4) when compared to control animals. Mice treated with AlCl3 (300 mg/kg; p.o.) for 1 month showed a significant increase in day 4 ELT values (Fig. 3) and decrease in day 5 TSTQ (Fig. 4) in comparison to normal control animals indicating impairment in learning and memory. Administration of diltiazem (20 and 40 mg/kg; p.o.)/ donepezil (0.1 mg/kg; i.p.) in AlCl3-treated mice exhibited a
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Fig. 2 Effect of diltiazem on TLT of AlCl3-treated mice using elevated plus maze. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl 3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus day 1 TLT in control group; bp < 0.05 versus day 2 TLT in control group; c p < 0.05 versus day 2 TLT in AlCl3-treated group
DAY 4 ELT (sec)
Fig. 3 Effect of diltiazem on day 4 ELT of AlCl3-treated mice using Morris water maze. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.), D.H2O distilled water. Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus day 1 ELT in control group; bp < 0.05 versus day 4 ELT in control group; cp < 0.05 versus day 4 ELT in AlCl3-treated group
significant fall in day 4 ELT (Fig. 3) and rise in day 5 TSTQ (Fig. 4) indicating reversal of learning and memory. Furthermore, diltiazem per se and donepezil per se did not show any significant effect on day 4 ELT (Fig. 3) and day 5 TSTQ (Fig. 4). Effect on brain AChE activity AlCl3-treated mice showed a significant rise in brain AChE activity when compared to the control group (Fig. 5). Administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg; i.p.) prevented AlCl3-induced increase in brain AChE activity (Fig. 5). However, diltiazem per se, donepezil per se, and vehicle groups did not show any effect on brain AChE activity (Fig. 5). Effect on brain TBARS level AlCl3-treated mice showed a significant increase in brain TBARS level when compared to the control group (Fig. 6). Administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg; i.p.) prevented AlCl3-induced increase in brain TBARS level (Fig. 6). Furthermore, diltiazem per se, donepezil per se, and vehicle groups did not show any effect on brain TBARS level (Fig. 6). Effect on brain reduced GSH level AlCl3-treated mice showed a significant decline in brain reduced GSH level in comparison with control group animals (Fig. 7). Administration of diltiazem (20 and 40 mg/kg; p.o.)/donepezil (0.1 mg/kg, i.p.) prevented
Fig. 4 Effect of diltiazem on mean time spent in target quadrant (TSTQ) (Q4) of AlCl3-treated mice using Morris water maze (MWM) test. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.),
Mean Time Spent in Target Quadrant (sec)
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AlCl3-induced decrease in brain GSH level (Fig. 7). However, administration of diltiazem per se, donepezil per se, and normal saline did not show any significant changes in brain reduced GSH level when compared with control group animals (Fig. 7).
Effect on brain nitrite/nitrate level AlCl3-treated mice showed a significant rise in brain nitrite/ nitrate level when compared to the control group (Fig. 8). Administration of diltiazem (20 and 40 mg/kg; p.o.)/ donepezil (0.1 mg/kg; i.p.) prevented AlCl3-induced increase in brain nitrite/nitrate level (Fig. 8). Diltiazem per se, donepezil per se, and normal saline group did not show any significant effect on brain nitrite/nitrate level when compared to control group (Fig. 8). 200 AChE activity (nM/min/mg of protein)
Fig. 5 Effect of diltiazem on brain acetylcholinesterase (AChE) activity in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± Standard error of mean (SEM), n = 8, oneway ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus brain AChE activity in control, bp < 0.05 versus brain AChE activity in AlCl3-treated group
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Effect on brain superoxide dismutase activity AlCl3-treated mice showed a significant decrease in brain superoxide dismutase (SOD) activity in comparison to control group animals (Fig. 9). Administration of diltiazem (20 and 40 mg/kg, p.o.)/donepezil (0.1 mg/kg, i.p.) to AlCl3-treated mice showed a significant rise in brain superoxide dismutase activity (Fig. 9). However, administration of diltiazem per se, donepezil per se, and normal saline did not show any significant variation in brain superoxide dismutase activity when compared with normal control animals (Fig. 9).
Discussion In present study, MWM and elevated plus maze are used to evaluate learning and memory in experimental animals. In a b
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Brain TBARS (nmol/mg of protein)
Fig. 6 Effect of diltiazem on brain thiobarbituric acid-reactive species (TBARS) level in AlCl3treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus brain TBARS level in control; bp < 0.05 versus brain TBARS level in AlCl3-treated group
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MWM, fall in day 4 ELT and rise in day 5 TSTQ in search of platform indicate memory retrieval. In elevated plus maze, fall in day 2 TLT indicates normal learning and memory. The normal control mice showed decreased day 4 ELT and increased day 5 TSTQ using MWM indicating normal learning and memory. Further, control mice show decreased day 2 TLT in elevated plus maze indicating no impairment in memory. Administration of vehicle, i.e., normal saline, did not show any additional effect on memory retrieval. Furthermore, no per se effects are seen on acquisition and retrieval in comparison to the normal group. These results agree with the results of previous studies done in other laboratories (Kumar et al. 2011; Saraf et al. 2011). AlCl3 (300 mg/kg; p.o.) was administered for 30 days and resulted in memory impairment in mice as indicated by a significant increase in day 4 ELT and decrease in day 5 30 Brain GSH (micromoles/mg of protein)
Fig. 7 Effect of diltiazem on brain reduced glutathione (GSH) level in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, oneway ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus brain GSH level in control; bp < 0.05 versus brain GSH level in AlCl3-treated group
TSTQ. AlCl3 administration also increased day 2 TLT which also indicates impaired learning and memory. Moreover, AlCl3-treated animals showed a significant increase in brain AChE activity, brain nitrite/nitrate levels, and brain TBARS levels along with depletion of brain SOD and GSH levels. In the present study, a marked decrease in body weight in aluminum exposed animals was observed as compared to the animals of control group. These findings are also in line with previous studies done in other laboratories (Cherrot et al. 1995; Sharma et al. 2007). Aluminum is a ubiquitous metal that has been implicated in the etiology of neurodegenerative disorders where it exacerbates brain oxidative damage, neuroinflammation, and Aβ deposition (Becaria et al. 2002). Aluminum exposure exerted adverse effects on learning and memory which were manifested by increase in number of acquisition and
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Naunyn-Schmiedeberg's Arch Pharmacol 25 Brain Nitrite/Nitrate (µg/mg of protein)
Fig. 8 Effect of diltiazem on brain nitrite/nitrate level in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, one-way ANOVA followed by Tukey’s multiple range test. ap < 0.05 versus brain nitrite/nitrate level in control; b p < 0.05 versus brain nitrite/ nitrate in AlCl3-treated group
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retention errors of rodents. Aluminum causes disturbances in cholinergic neurotransmission and disrupts the cognitive behavior of animals by increasing the stress levels. The elevations in AChE are either direct result of neurotoxic effect of metals or due to increased lipid peroxidation (Kaizer et al. 2008). Studies have shown that aluminum exposure results in decrease of hexokinase activity, which in turn decreases the pyruvate formation and hence affects the synthesis of acetylcholine and subsequently the enzyme activity of AChE. Aluminum is a pro-oxidant and indirectly results in the production of free radicals leading to oxidative damage and reduced levels of ROS which indirectly affect acetylcholinesterase enzyme activity (Yuan et al. 2012). Aluminum causes marked oxidative damage by increasing the redox active iron concentration in the brain mainly via the Fenton reaction (Exley 2004). Aluminum causes reduced axonal mitochondria
turnover, disruption of Golgi, and reduction of synaptic vesicles which result in release of oxidative products like malondialdehyde, carbonyls, peroxynitrites, and enzymes like SOD within the neurons (Yuan et al. 2012). Aluminum can also induce neurotoxicity via impairing neuronal exchange of calcium ions and increase Ca2+ level in brain (Goma and Mahrous 2013). The increased Ca2+ causes mitochondrial overload, excessive production of Aβ, and tau hyperphosphorylation which further generate caspases and cytochrome c and increase ROS production leading to cell death and neurodegeneration (Stutzmann 2007). Administration of diltiazem (20 and 40 mg/kg p.o. for 14 days) to AlCl3-treated mice results in decreased day 4 ELT and increased day 5 TSTQ which indicate improved learning and memory. Diltiazem administration also showed a significant decrease in day 2 TLT. Further, diltiazem reduces
60 SOD activity (picomole/min/mg og wet tissue)
Fig. 9 Effect of diltiazem on brain superoxide dismutase (SOD) activity in AlCl3-treated group. DTZ (HD) diltiazem high dose (40 mg/kg; p.o.), DTZ (LD) diltiazem low dose (20 mg/kg; p.o.), AlCl3 aluminum chloride (300 mg/kg; p.o.), Don donepezil (0.1 mg/kg; i.p.). Values are expressed as mean ± standard error of mean (SEM), n = 8, oneway ANOVA followed by Tukey’s multiple range test. a p < 0.05 versus brain SOD activity in control; bp < 0.05 versus brain SOD activity in AlCl3-treated group
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brain AChE, brain TBARS, and brain nitrite/nitrate levels and increases brain GSH and SOD activity. Diltiazem administration also improved the body weight in AlCl3-treated mice. Moreover, administration of donepezil (0.1 mg/kg; i.p. for 14 days) attenuates the brain changes induced by administration of AlCl3. These results are consistent with the results of previous studies (Packard et al. 1996). In the present study, donepezil has been used as a positive control. Diltiazem, a CCB drug, has been evidenced to be useful in hypertension, angina, and cardiovascular disease. Diltiazem has vasodilatory effects on arterial vessels and it increases the reflex in sympathetic response. Its overall effect in CVD is a drop in blood pressure and an increase in cardiac output, heart rate and contractility (Gura 2008). Other than CVD, diltiazem has also been suggested to be useful in general cognitive enhancement on the basis of its ability to improve learning and memory in adult animals (Quartermain et al. 2001). The concentrations (dose) employed in the study were chosen after performing the pilot studies starting with very low dose and referring to the previous reports. A range of concentrations have been used in animals varying from as low as 5 mg/kg to as high as 1000 mg/kg for 40–60 days (Biala and Kruk 2009; Bishnoi et al. 2008; Correa et al. 2009). Voltage-gated calcium channels play an important role in the entry of Ca2+ ions into excitable cells and are also involved in a series of calcium-dependent processes, such as muscle contraction, hormone or neurotransmitter release, gene expression, and cell death (Augustine et al. 1987; Miller 1987). Voltage-gated calcium channels are blocked by 1,4dihydropyridines (DHPs) such as nifedipine, phenylalkylamines such as verapamil, and benzothiazepines such as diltiazem (Shaldam et al. 2014). They are multisubunit complexes, comprising alpha-1, alpha-2, beta, and delta subunits. The channel activity is directed by the pore-forming and voltage-sensitive alpha-1 subunit. In many cases, this subunit is sufficient to generate voltage-sensitive calcium channel activity that displays the major electrophysiological and pharmacological properties of the full-fledged heteromeric channels (Soldatov et al. 1995; Zuhlke et al. 1998). It has been proposed to be useful in Alzheimer’s disease because of its calcium-lowering effect across the voltage-gated channels. Increased calcium levels are postulated in increased mitochondrial overload, oxidative damage, Aβ production, and tau phosphorylation (Iqbal and Grundke 2007). Increased Aβ further interacts with Fe2+ and Cu2+ to generate ROS, and ROS leads to lipid peroxidation (Zhu et al. 2006). Diltiazem thus, by blocking Ca2+ effects, avoids these adverse effects of calcium dysregulation. Furthermore, diltiazem also showed antiinflammatory effects by stimulating production of antiinflammatory mediators IL-10 and inhibiting proinflammatory mediator IL-6 (Dubey and Hesong 2006). Even though, numerous studies have suggested the neuroprotective potential of calcium channel blockers in disorders such
as parkinsonism (Ritz et al. 2010), Alzheimer’s disease (Anekonda and Quinn 2011), focal cerebral ischemia (Takahara et al. 2004) and spinal cord ischemia-reperfusion injury (Fansa et al. 2009). However, till date, none of the reports suggest the memory restorative ability of diltiazem. This is the first report in itself that speculates the protective role of diltiazem in memory and learning, although this also necessitates in-depth evaluation of the exact mechanism of diltiazem in dementia. Thus, from the literature survey and data in hand, it may be concluded that diltiazem has a neuroprotective effect on AlCl3-induced behavioral and biochemical parameters in mice.
Conclusion The results obtained from our study indicate that diltiazem significantly improves AlCl3-induced memory impairment and biochemical changes. This is the first report indicating the memory-restorative ability of a calcium channel blocker in metal-induced dementia. The improvement in learning and memory produced may be owed to its anti-oxidative and anticholinesterase potential. However in-depth in vitro and in vivo studies need to be performed to evaluate the exact mechanism of diltiazem in memory enhancement.
Acknowledgments The authors are thankful to Chandigarh College of Pharmacy, Landran (India) for supporting this study and providing technical facilities for the work.
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