580 Research report
FK506 attenuates intracerebroventricular streptozotocininduced neurotoxicity in rats Rimpi B. Arora, Kushal Kumar and Rahul R. Deshmukh Upregulation in calcineurin (CaN) signaling has been implicated in various neurodegenerative disorders. In the present study, we have investigated the effect of FK506 – a CaN inhibitor – on streptozotocin (STZ)induced experimental dementia of the Alzheimer’s type in rats. STZ was administered intracerebroventricularly to induce a cognitive deficit and oxidative stress. Nonimmunosuppressive doses (0.5 and 1 mg/kg postoperatively) of FK506 (tacrolimus) were administered for 21 day in STZ-treated rats. Cognitive functions were assessed using the Morris water maze and passive avoidance tasks. Malondialdehyde and nitrite glutathione levels, as well as acetylcholinesterase activity, were determined to evaluate oxidative stress and cholinergic functions. Lactate dehydrogenase levels were estimated and histological analysis of the dentate gyrus and the CA1 region of the hippocampus was carried out to identify degenerative changes. STZ produced significant deterioration of cognitive functions, oxidative stress, and degenerative changes in the cortical and hippocampal
brain regions. FK506 dose-dependently attenuated STZ-induced cognitive deficits, oxidative stress, and degenerative changes in the cortex and hippocampus. These results suggest a potential role of CaN signaling in degenerative processes, and that inhibition of CaN may be useful in the treatment of neurodegenerative disorders such as Alzheimer’s disease. Behavioural c 2013 Wolters Kluwer Health | Pharmacology 24:580–589 Lippincott Williams & Wilkins.
Introduction
2008), Huntington’s disease (Xifiro et al., 2008; Dudek et al., 2010), and AD (Dineley et al., 2007).
Alzheimer’s disease (AD) is an age-related neurodegenerative disorder (Reed et al., 2009). Accumulating data indicate that disturbances in several aspects of cellular metabolism appear pathologically important in AD. Among these, increased brain insulin resistance (Watson and Craft, 2006; Hoyer and Lannert, 2007; SalkovicPetrisic, 2008) and decreased glucose utilization (Balbo et al., 2002; Steen et al., 2005; De la Torre, 2008) and energy metabolism are observed in the early stages of the disease (De la Monte and Wands, 2005; Hoyer and Lannert, 2007; Salkovic-Petrisic and Hoyer, 2007). Decreased energy metabolism has also been implicated in the activation of numerous signaling pathways and Ca2 + toxicity (Muller et al., 1998). One intracellular signaling element that has recently attracted attention as a potential modulator of both learning and memory function, as well as cell degeneration, is the Ca2 + /calmodulin-dependent protein serine– threonine phosphatase 2B, also known as calcineurin (CaN), which is the most abundant phosphatase in the brain (Mansuy, 2003; Liu et al., 2007) and is activated following Ca2 + influx (Liu, 2009). CaN has been implicated in the pathogenesis of various diseases such as cardiac hypertrophy (Nakamura et al., 2008), congenital heart disease (Groetzner et al., 2005), epilepsy (VazquezLopez et al., 2006), Parkinson’s disease (Wright et al., c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins 0955-8810
Behavioural Pharmacology 2013, 24:580–589 Keywords: cognitive dysfunction, FK506, oxidative stress, rat, sporadic dementia, streptozotocin, tacrolimus Department of Pharmacology, Neuropharmacology Division, ISF College of Pharmacy, Moga, Punjab, India Correspondence to Rahul R. Deshmukh, MPharm, PhD, Department of Pharmacology, Neuropharmacology Division, ISF College of Pharmacy, Moga 142001, Punjab, India E-mail: [email protected] Received 22 January 2013 Accepted as revised 4 July 2013
CaN has been reported to negatively regulate learning and memory (Eto et al., 2008). Inhibition of CaN signaling has been reported to improve associative learning and memory in Tg2876 APP transgenic mice (Dineley et al., 2007) and spatial memory in 3-nitropropionic acidinduced neurotoxic insult (Kumar and Kumar, 2009). CaN is specifically inhibited by certain drugs such as cyclosporine-A and FK506 in the presence of cyclophilin and FK506-binding protein (FKBP-12 and FKBP-52), respectively, resulting in immunosuppressant activity (Van Rossum et al., 2009). In addition, nonimmunosuppressive doses of FK506 have also been demonstrated to provide neuroprotection without affecting inflammatory mechanisms in various models such as experimental stroke (Butcher et al., 1997), spinal-cord injury (Voda et al., 2005), Parkinson’s disease (Wright et al., 2008) and Huntington’s disease (Kumar and Kumar, 2009). Despite these findings, the role of CaN and beneficial effects of FK506 in experimental sporadic dementia of Alzheimer’s type remain elusive. Intracerebroventricular administration of streptozotocin (ICV-STZ) in rats is commonly used to study experimental sporadic dementia of the Alzheimer’s type. ICV-STZ has been reported to produce behavioral, neurochemical, DOI: 10.1097/FBP.0b013e32836546db
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FK506 against STZ-induced neurotoxicity Arora et al. 581
biochemical, and histopathological changes similar to those in an aging brain and is considered to be a suitable animal model of sporadic dementia of the Alzheimer’s type (Prickaerts et al., 1995; Sharma and Gupta, 2001, 2002; Salkovic-Petrisic, 2008; Ishrat et al., 2009). ICV-STZ administration has also been reported to increase the level of cytoplasmic calcium ions (Muller et al., 1998), and the pharmacological blockade of L-type calcium channels (lercanidipine) has been reported to attenuate behavioral and biochemical alterations in ICV-STZ-injected rats (Sonkusare et al., 2005). Dysregulation in Ca2 + homeostasis has been observed in neurodegenerative disorders including AD. Moreover, dysregulation of calcium signaling has been reported to upregulate CaN signaling mechanisms (Foster et al., 2001), which may perhaps contribute to the degenerative changes and subsequent cognitive decline associated with sporadic AD. Thus, in the present study, we have investigated the potential effect of FK506, a CaN inhibitor, on STZ-induced neurotoxicity.
Methods Subjects
The experiments were carried out on male Wistar rats (220–250 g) procured from the Indian Institute of Integrative Medicine, Jammu, India. The animals were kept in polyacrylic cages and maintained under standard housing conditions (room temperature 22±21C and relative humidity of 60–65%), with lights on at 07.00 h and off at 19.00 h, at the ‘Central Animal House’ of ISF College of Pharmacy, Moga, Punjab (India). Food, in the form of dry pellets, and water were freely available. All behavioral experiments were carried out between 10.00 and 16.00 h. The protocol was reviewed and approved by the ‘Institutional Animal Ethics Committee’, and the animal experiments were carried out in accordance with the Indian National Science Academy Guidelines for use and care of animals. Intracerebroventricular infusion of streptozotocin
Rats were anesthetized with ketamine (100 mg/kg, intraperitoneally) and xylazine (5 mg/kg, intraperitoneally). The head was placed in position in the stereotaxic apparatus and a midline saggital incision was made in the scalp. Two holes were drilled through the skull for placement of infusion cannulae into the lateral cerebral ventricles using the following coordinates: 0.8 mm posterior to bregma; 1.5 mm lateral to the saggital suture; 3.6 mm ventral from the surface of the brain (Paxinos and Watson, 1986). After cannula placement, animals were injected with gentamicin (5 mg/kg) and were placed in individual cages. Animals were observed for 1 week, and special care was taken by administering sweetened milk daily during this resting phase for recovery (Fig. 1). After the resting period, STZ was dissolved in citrate buffer (pH 4.4) and slowly infused (1 ml/min), using a Hamilton microsyringe pump (Quintessential stereotaxic injector; Stoelting Co., Wood Dale, Illinois, USA), into each
cerebral ventricle (bilateral ICV) in a volume of 10 ml on days 1 and 3 (Deshmukh et al., 2009). Experimental groups
Animals were divided into five groups, each comprising 10 animals. The treatment schedule and the interval for estimation of various parameters are presented in Fig. 1. Group1, which served as a double-vehicle control, received citrate buffer (pH 4.4) ICV at a volume of 10 ml in each ventricle on days 1 and 3 and 0.5% carboxymethyl cellulose (0.5 ml intraperitoneally, as a vehicle for FK506) for 21 days. The animals in group 2 were treated with 1 mg/kg FK506 postoperatively. Rats in group 3 were infused with ICV-STZ at a dose of 3 mg/kg. Groups 4 and 5 received FK506 at doses of 0.5 and 1 mg/kg, respectively, postoperatively, 1 h after STZ administration; this was started on the first post operative day and continued once daily for a period of 21 days. The nonimmunosuppressive doses of FK506 were selected on the basis of earlier reports in which significant antioxidant and neuroprotective properties were demonstrated at doses ranging from 0.2 to 3 mg/kg (Sakai et al., 1991; Singh et al., 2003a, Gold et al., 2004; Wright et al., 2008). Behavioral assessment Passive avoidance task
On days 14 and 15 after ICV-STZ infusion, the rats were tested for memory retention using a passive avoidance apparatus. The apparatus (Type-7552; Ugo Basile, Comerio, Italy) consisted of a chamber illuminated with a 40-W bulb and a dark chamber, separated by a guillotine door. The floor of the dark chamber consisted of a metal grid with a shock scrambler. During the acquisition trial, the rat was placed in the illuminated chamber. After an initial habituation period of 60 s, the guillotine door was opened and the time taken by the rat to enter the dark chamber was noted. The latency to step into the dark compartment was recorded as the initial trial latency (ITL) or preshock latency. As soon as the rat entered the dark chamber, it was given a mild foot shock of 0.5 mA for 2 s through the grid floor. The rat was allowed to remain in the dark compartment for 5 s and then taken out. After a 24-h interval, a retention trial was performed and retention trial latency (RTL) or postshock latency to step into the dark compartment was noted. The latency was recorded to a maximum of 600 s (Deshmukh et al., 2009). Short latencies indicated poorer retention. Morris water maze
Spatial learning and memory were tested in a Morris water maze (MWM; Morris, 1984), which consisted of a circular water tank (180 cm diameter, 60 cm height) filled with water (25±11C) to a depth of 40 cm. Milk powder was used to render the water opaque. Four equally spaced locations around the edge of the pool (North, South, East, and West) were used as start points, which divided
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Fig. 1
CI
Rest
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ICV-STZ Day1
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Day 3
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Day 21
Tacrolimus (0.5 and 1 mg/kg, per oral) LA
SAC
Experimental procedure and treatment schedule. CI, cannula implantation; ICV, intracerebroventricular; LA, locomotor activity; MWM, Morris water maze; PAL, passive avoidance latency; SAC, sacrificed; STZ, streptozotocin.
the pool into four quadrants. An escape platform (10 cm in diameter) was placed 2 cm below the water surface in the middle of the north-east quadrant of the pool and kept in the same position throughout the experiment. Before training started, the rats were allowed to swim freely in the pool for 120 s without a platform. Animals underwent a training session consisting of four trials per session (once from each starting point) for 4 days (days 17, 18, 19, and 20), each trial having a ceiling time of 120 s and an intertrial interval of B30 s. After climbing onto the hidden platform, the animals were allowed to remain there for 30 s before commencement of the next trial. If the rat failed to locate the hidden platform within the maximum time of 120 s, it was gently placed onto the platform and allowed to remain there for the same interval of time. Twenty-four hours after the acquisition phase, a probe test (day 21) was conducted by removing the platform. Rats were allowed to swim freely in the pool for 60 s and the percentage of time spent in the target quadrant, which had previously contained the hidden platform, was recorded. The time spent in the target quadrant indicated the degree of memory consolidation that had taken place after learning (Deshmukh et al., 2009). The time taken to locate the hidden platform and the time spent in the target quadrant during the probe trial was measured by an experimenter who was blind to treatment, using a stopwatch. Spontaneous locomotor activity
Each animal was tested for spontaneous locomotor activity on day 21 after the first ICV-STZ infusion. Each animal was observed over a period of 10 min in a square closed arena equipped with infrared light sensitive photocells, using a digital photoactometer (INCO, Ambala, Haryana, India) (Deshmukh et al., 2009). Biochemical parameters Brain homogenate preparation
All biochemical parameters were measured in brain homogenates. On day 22 after the first STZ infusion, animals were killed by decapitation and brains were removed and rinsed with ice-cold isotonic saline. Brain
tissue samples were then homogenized with ice-cold 0.1 mol/l phosphate buffer (pH 7.4) in a volume 10 times the weight of the tissue. The whole brain homogenate was centrifuged at 10 000g for 15 min and aliquots of the supernatant were separated and used for biochemical estimation. Protein estimation
Protein levels in all brain samples were estimated using the method developed by Lowry et al. (1951), with BSA (1 mg/ml) as a standard. Acetylcholinesterase assay
The quantitative measurement of acetylcholinesterase (AChE) activity in the brain was performed according to the method described by Ellman et al. (1961). The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 mol/l sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide, and 0.10 ml of 5,50 -dithiobis(2nitrobenzoic acid) (Ellman reagent). The change in absorbance was measured immediately at 412 nm spectrophotometrically. AChE activity in the supernatant was expressed as nmol/mg protein. Estimation of malondialdehyde
The quantitative measurement of malondialdehyde (MDA), the end product of lipid peroxidation, was performed according to the method developed by Wills (1966). The amount of MDA was measured after its reaction with thiobarbituric acid at 532 nm, using a spectrophotometer (UV-1700; Shimadzu, Columbia, Maryland, USA). The concentration of MDA was determined from a standard curve and expressed as nmol/mg protein. Estimation of reduced glutathione
Reduced glutathione (GSH) levels in the brain were estimated according to the method described by Ellman (1959). One milliliter of supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 41C for 1 h. The samples were centrifuged at 1200g for 15 min. To 1 ml of the supernatant, 2.7 ml of phosphate buffer (0.1 mol/l, pH 8) and 0.2 ml of DTNB were added. The yellow color that developed was measured immediately at 412 nm using a spectrophotometer.
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FK506 against STZ-induced neurotoxicity Arora et al. 583
The concentration of GSH in the supernatant was determined from a standard curve and expressed as mmol/mg protein.
prepared afresh by suspending in 0.5% sodium carboxymethyl cellulose. All other chemicals used in the study were of analytical grade. Solutions of the drug and chemicals were freshly prepared before use.
Estimation of nitrite
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined by a colorimetric assay using the Greiss reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid], as described by Green et al. (1982). Equal volumes of the supernatant and Greiss reagent were mixed, the mixture was incubated for 10 min at room temperature in the dark, and the absorbance was determined at 540 nm spectrophotometrically. The concentration of nitrite in the supernatant was determined from the sodium nitrite standard curve and expressed as mmol/mg protein.
Statistical analysis
Assessment of histological changes
On day 14 after the first ICV-STZ injection, the mean initial latency in the acquisition trial remained unchanged among all the groups [F(4,49) = 2.21, NS]. However, the mean retention latency on day 15 was significantly decreased (P < 0.001) in the ICV-STZ control group compared with vehicle-treated animals (Fig. 2). The ICV-STZ-induced decrease in the mean retention latency was significantly increased on FK506 treatment [F(4,49) = 174.3, P < 0.001], indicating protection against the effect of STZ treatment. The maximum improvement in retention latency by FK506 treatment on day 15 was observed at a dose of 1 mg/kg (P < 0.001). Moreover, control rats treated with FK506 (1 mg/kg) alone did not show any significant difference in retention latency as compared with vehicle-treated rats (P > 0.05).
The brains were rapidly removed and fixed by immersion in 10% formalin. Subsequently they were embedded in paraffin wax, cut into 5-mm-thick sections, and stained with cresyl violet acetate (Nissl stain; Shoham et al., 2003). Hippocampal brain sections were examined under bright field illumination (AHBT-51, Olympus VanoxAHBT; Olympus America, Melville, New York, USA). Drugs
STZ, acetylthiocholine iodide, and DTNB were purchased from Sigma-Aldrich (St Louis, Missouri, USA). FK506 was provided as an ex-gratia sample by M/S Panacea Biotec Ltd. (Mohali, Punjab, India). STZ was diluted in citrate buffer (pH 4.4) and FK506 was always
The results are expressed as mean±SD. The behavioral (for individual time points) and biochemical values were analyzed by one-way analysis of variance, followed by Tukey’s post-hoc test for multiple comparisons, using GraphPad Prism statistical software (version 5.0; GraphPad Prism software Inc., La Jolla, California, USA). A P-value of less than 0.05 was set to be statistically significant.
Results Effect of FK506 on memory performance in the passive avoidance task in ICV-STZ-treated rats
Fig. 2
Transfer latency (s)
600
Day-14 aquisition trial
Day-15 retention trial
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m (1 FK
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Effect of FK506 on performance of ICV-STZ-treated rats in the passive avoidance task. Values are expressed as mean±SD; @P < 0.05 versus vehicle; *P < 0.05 versus STZ; #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
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584 Behavioural Pharmacology 2013, Vol 24 No 7
Effect of FK506 on memory performance in the MWM task in ICV-STZ-treated rats
Effect of FK506 on brain GSH levels in ICV-STZ-treated rats
On day 17 of the acquisition trial, experimental animals from all the groups took a similar amount of time to locate the hidden platform in the MWM task [F(4,49) = 1.109, P > 0.05], but on days 18, 19, and 20 of the acquisition phase, STZ-injected rats showed a significant impairment in learning relative to the vehicle control group [F(4,49) = 59.07, 34.12, and 71.83, respectively, P < 0.001] (Fig. 3a). Moreover, during the probe trial [F(4,49) = 39.64, P < 0.01], STZ-treated rats also failed to remember the precise location of the platform, spending significantly less time in the target quadrant as compared with vehicletreated animals (P < 0.001; Fig. 3b). Chronic administration of FK506 in STZ-treated rats significantly and dosedependently attenuated STZ-induced acquisition and retention deficits in the MWM task. In the probe trial, FK506-treated rats spent more time in the target quadrant, as compared with STZ-treated control rats, indicating improved consolidation of memory (P < 0.05).
STZ administration in rats produced a significant depletion in GSH levels in the brain [F(4,49) = 83.90, P < 0.001] (Table 1) as compared with vehicle treatment (P < 0.001). Chronic administration of FK506 significantly restored GSH levels in STZ-treated rats (P < 0.01) without affecting GSH levels in control rats.
Effect of FK506 on spontaneous locomotor activity in ICV-STZ-treated rats
Effect of FK506 on the neuronal morphology of the dentate gyrus and the CA1 region of the hippocampus in ICV-STZ-treated rats
Spontaneous locomotor activity on day 21 did not differ significantly among groups [F(4,49) = 0.41, NS] (Fig. 4). Effect of FK506 on brain AChE activity in ICV-STZtreated rats
Ventricular administration of STZ produced a significant increase in brain AChE activity relative to vehicle controls [F(4,34) = 63.28, P < 0.001] (Fig. 5). Chronic administration of FK506 (0.5 and 1 mg/kg) dose-dependently attenuated the STZ-induced increase in AChE activity (P < 0.01). However, FK506 did not affect AChE activity in control rats (P > 0.05). Effect of FK506 on MDA levels in ICV-STZ-treated rats
Administration of STZ in rats produced a pronounced elevation in MDA levels, as compared with vehicle treatment [F(4,34) = 92.38, P < 0.001] (Table 1). Chronic administration of FK506 dose-dependently attenuated the STZ-induced elevation in MDA levels (P < 0.001). However, FK506 treatment did not produce any significant effect in normal animals when compared with vehicle controls (P > 0.05). Effect of FK506 on brain nitrite level in ICV-STZ-treated rats
STZ-treated rats showed a significant increase in brain nitrite levels as compared with vehicle control-treated animals [F(4,34) = 98.44, P < 0.001] (Table 1). The STZinduced increase in nitrite levels was significantly and dose-dependently attenuated by FK506 (0.5 and 1 mg/kg, P < 0.01), whereas FK506 administration did not produce any significant effect (P > 0.05) in control animals as compared with vehicle-treated animals.
Effect of FK506 on brain lactate dehydrogenase levels in ICV-STZ-treated rats
STZ produced a significant elevation in brain lactate dehydrogenase (LDH) levels [F(4,34) = 59.67, P < 0.001] (Fig. 6) as compared with vehicle treatment, indicating cellular damage. FK506 (0.5 and 1 mg/kg) dose-dependently attenuated the STZ-induced elevation in LDH levels (P < 0.01). However, FK506 treatment did not alter LDH levels in the brains of normal rats as compared with vehicle treatment (P > 0.05).
Cresyl violet-stained hippocampal dentate gyrus and CA1 sections of vehicle-treated animals showed healthy neurons (Fig. 7a, b), which were robust in shape, had a pale and spherical or slightly oval nucleus, and had a single large nucleolus with a clear, visible cytoplasm. Brain sections from rats treated with FK506 (1 mg/kg, postoperatively) also showed healthy neurons in the dentate gyrus and CA1 region (Fig. 7c, d). Similar morphology and neuronal density in the dentate gyrus and the hippocampal CA1 region were observed in the vehicle-treated and FK506-treated groups (Fig. 7a–d). Neuronal degeneration may present a continuum of neurodegenerative morphologies. In this study, brain sections from ICV-STZ-treated rats showed pyknotic neurons that were darkly stained with no nucleus or visible nucleolus, and a few of the cells were shrunken and sickle shaped. Photomicrographs of the dentate gyrus and CA1 region of the hippocampus of ICV-STZ-treated groups showed a marked decrease in cell bodies (Fig. 7e, f). Other neurons showed a smaller nucleus with condensed chromatin and a swollen cytoplasm. Further, STZ administration markedly decreased neuronal density and increased the number of pyknotic neurons. Chronic treatment with FK506 (0.5 mg, Fig. 7g, h; 1 mg, Fig. 7i, j) attenuated STZ-induced cell loss and pyknosis; however, some degenerating cells with changes in morphology were observed in the FK506 treatment groups. Sections from rats treated with the higher dose of FK506 showed rounded and open nuclei with marked protection from STZ-induced neurodegenerative morphologies (Fig. 7i, j). There was marked improvement in neuronal density and reduction in pyknotic neurons following chronic FK506 treatment in STZ-treated rats, indicating a neuroprotective potential, in line with behavioral and biochemical effects of FK506.
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FK506 against STZ-induced neurotoxicity Arora et al. 585
Fig. 3
(a)
120
Vehicle
FK per se
STZ
FK (0.5 mg)
FK (1 mg)
Mean escape latency (s)
100 @
80
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@
@
∗# 60
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Time spent in target quadrant (%)
(b)
Day 18
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FK (0.5 mg)
FK (1 mg)
Effect of FK506 on performance of ICV-STZ-treated rats in the Morris water maze. (a) Escape latency and (b) time spent in the target quadrant. Values are expressed as mean±SD; @P < 0.05 versus vehicle, *P < 0.05 versus STZ, #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin. Effect of tacrolimus on time spent in target quadrant in ICV-STZ-treated rats is expressed as mean±SD. @ P < 0.05 versus Vehicle, *P < 0.05 versus STZ, #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
Discussion The present study demonstrated a neuroprotective role of FK506, a specific CaN inhibitor, against STZ-induced behavioral, biochemical, and histological toxicity in rats. In line with earlier reports, ICV-STZ was found to produce significant impairment in spatial cognitive functions and increase oxidative–nitrosative stress (Sharma and Gupta, 2001; Sonkusare et al., 2005; Ishrat et al., 2006; Salkovic-Petrisic and Hoyer, 2007; SalkovicPetrisic, 2008). FK506 dose-dependently (0.5 and 1 mg/kg postoperatively) protected against the effect of STZ treatment in the MWM and passive avoidance task (Fig. 2 and Fig. 3a, b) without affecting locomotor activity (Fig. 4). Changes in locomotor activity have been suggested to modulate learning and memory in the passive avoidance and MWM paradigms (Sharma and
Gupta, 2003; Deshmukh et al., 2009). However, no significant difference in spontaneous locomotor activity was observed in any of the experimental groups in the present study. The cholinergic system plays an important role in memory formation and retrieval (Olton, 1990; Fibiger, 1991; Fibiger et al., 1991; Blokland, 1995). In the present study, STZ-induced cognitive deficit was also associated with an increase in cholinesterase activity (Fig. 5), which is in accordance with the findings of earlier studies (Sonkusare et al., 2005; Ishrat et al., 2006; Deshmukh et al., 2009). In the present study, FK506 dose-dependently protected against the effect of STZ treatment in the MWM and passive avoidance task and restored cholinesterase activity without modifying basal memory and cholinesterase
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586 Behavioural Pharmacology 2013, Vol 24 No 7
Fig. 4
Fig. 6
500 LDH levels (IU/mg protein)
Activity counts/10 min
300
200
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0
@
400 300
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FK per se
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Effect of FK506 on spontaneous locomotor activity in ICV-STZ-treated rats. Values are expressed as mean±SD. FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
Vehicle
FK per se
STZ
FK 0.5 mg
FK 1 mg
Effect of FK506 on brain lactate dehydrogenase in ICV-STZ-treated rats. Values are expressed as mean±SD; @P < 0.05 versus vehicle; *P < 0.05 versus STZ; #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
Fig. 5
Acetylcholinesterase activity (nmol/mg protein)
500
@
400 ∗ 300
∗#
200 100 0 Vehicle
FK per se
STZ
FK (0.5 mg) FK (1 mg)
Effect of FK506 on brain acetylcholinesterase activity in ICV-STZtreated rats. Values are expressed as mean±SD; @P < 0.05 versus vehicle; *P < 0.05 versus STZ; #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, Intracerebroventricular; LDH, lactate dehydrogenase; STZ, streptozotocin.
Table 1
Effect of FK506 on biochemical parameters Biochemical parameters
Groups Vehicle FK per se STZ FK (0.5 mg) FK (1 mg)
MDA (nmol/mg protein)
GSH (mmol/mg protein)
Nitrite (mmol/mg protein)
168.6±16.57 163.2±27.44 411.2±40.71@ 278.4±27.75* 213.6±23.32*#
1.84±0.23 1.95±0.28 0.18±0.08@ 0.91±0.18* 1.31±0.21*#
0.43±0.25 0.42±0.18 3.47±0.54@ 2.20±0.33* 1.55±0.28*#
Values are expressed as mean±SD. FK, FK506; GSH, reduced glutathione; MDA, malondialdehyde; STZ, streptozotocin. @ P < 0.05 versus vehicle. *P < 0.05 versus STZ. # P < 0.05 versus FK506 (0.5 mg/kg).
activity. CaN is a Ca2 + /calmodulin-dependent protein serine–threonine phosphatase 2B and the most abundant phosphatase in the central nervous system (CNS)
(Mansuy, 2003; Liu et al., 2007). Impairment of insulin signaling and glucose and energy metabolism following administration of STZ into the CNS has been suggested to cause Ca2 + overactivation (De la Monte and Wands, 2005; Sonkusare et al., 2005), which may lead to further imbalance between kinases and phosphatases (SalkovicPetrisic and Hoyer, 2007; Salkovic-Petrisic, 2008). Moreover, it has been reported that CaN is the first among the postsynaptic phosphatases to be activated in response to Ca2 + influx (Liu, 2009). Indeed, ICV-STZ administration has been reported to increase intracellular Ca2 + concentration (Muller et al., 1998), and blockade of the L-type calcium channel by lercanidipine has been reported to improve cholinergic function and cognitive behavior in STZ-treated rats (Sonkusare et al., 2005). In the present study, STZinduced cognitive deficits and the increase in AChE levels were significantly attenuated by FK506. It can be speculated that the increase in intracellular Ca2 + concentration following STZ administration may subsequently lead to CaN overactivation and contributes to cognitive deficit. Indeed, overactivation of CaN has been reported to negatively regulate learning and memory (Mansuy, 2003; Baumgartel et al., 2008). In addition, acute inhibition of CaN by FK506 has been reported to restore associative learning and memory in Tg2576 APP mice (Dineley et al., 2007; Taglialatela et al., 2009). Oxidative stress has been implicated in the pathogenesis of AD in humans (Pratico, 2008; Mangialasche et al., 2009). Oxyradical-induced damage to macromolecules (lipids, proteins, and nucleic acids, etc.) is considered an important factor in the acceleration of aging and agerelated neurodegenerative disorders such as AD (Liu et al., 2001; Wickens, 2001). An STZ-induced impairment in cerebral energy metabolism and mitochondrial disturbances may be responsible for increased intracellular Ca2 + influx and generation of reactive oxygen species,
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FK506 against STZ-induced neurotoxicity Arora et al. 587
Fig. 7
(a, c, e, g, i) Cresyl violet-stained sections of the dentate gyrus and (b, d, f, h, j) the CA1 region of the hippocampus following treatment with (a, b) vehicle, (c, d) FK506 (1 mg/kg), (e, f) ICV-STZ, and chronic treatment with (g, h) 0.5 mg FK506 and (i, j) 1 mg FK506 following ICV-STZ treatment. (d, f, h, j) Pictures taken at a magnification of 400. FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
leading to oxidative stress (Muller et al., 1998; Shoham et al., 2007; Deshmukh et al., 2009). Further, ICV administration of STZ has been reported to cause Ca2 + -dependent production of NO, a precursor for free radicals, which reacts with superoxide anions to form peroxynitrite, causing nitrosative stress (Shoham et al., 2007; Kumar and Kumar, 2009). Consistent with previous studies, in the present study, ICV-STZ treatment significantly increased MDA and nitrite levels and decreased the levels of endogenous antioxidant enzymes, indicating increased oxidative–nitrosative stress (Deshmukh et al., 2009). Chronic treatment of STZ-treated rats with FK506 dose-dependently attenuated the STZ-induced increase in oxidative–nitrosative stress. These results suggest that FK506 may have direct antioxidant potential
or may decrease free radical generation by inhibiting CaN activity. It has been reported that oxidative stress regulates Ca2 + dependent serine–threonine phosphatase, that is, CaN (Manik et al., 2003). In addition, mitochondrial dysfunction and oxidative stress have been observed in CaN transgenic mice, indicating that the upregulation of CaN signaling may lead to oxidative burden (Sayen et al., 2003). In addition, CaN has been reported to dephosphorylate nitric oxide synthase, resulting in an increase in NO production (Knirsch et al., 2001). Indeed, FK506, by CaN inhibition, has been reported to negatively regulate Ca2 + -channel activity and inhibit free radical generation and nitric oxide synthase activity
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588 Behavioural Pharmacology 2013, Vol 24 No 7
(Victor et al., 1995; Phillis et al., 2002; Sasaki et al., 2004). In addition, FK506 has also been reported to possess direct free radical scavenging properties and to improve antioxidant defense mechanisms (Maziere et al., 2005). Therefore, FK506 may produce its antioxidant effect directly through free radical scavenging or indirectly through CaN inhibition. In the brain, STZ has been reported to cause neurodegeneration accompanied by an increase in LDH activity (Hoyer and Lannert, 2007). Consistent with these findings, in the present study, ICV-STZ-treated animals showed a marked increase in LDH levels, indicating cell damage. However, chronic administration of FK506 significantly attenuated the STZ-induced elevation in LDH levels. In line with the biochemical alterations, histological sections of the dentate gyrus and the CA1 region of the hippocampus of STZ-treated rats showed decreased neuronal density and necrotic cell death, as evidenced by pyknotic nuclear changes. Chronic administration of FK506 dose-dependently attenuated STZ-induced neurotoxicity and was able to restore neuronal density and a normal morphological pattern (Fig. 7), suggesting a neuroprotective potential of FK506. Ca2 + mobilization following stimulation of N-methyl-Daspartate receptors by glutamate has been implicated in cell death in the CNS. The Ca2 + sensitivity of nitric oxide synthase makes the Ca2 + -dependent phosphatase, CaN, an obvious candidate that affects neuronal cell death (Dawson et al., 1993), and therefore, CaN may be a material factor in Ca2 + -activated neuronal cell death (Shibasaki and Mckeon, 1995; Norris et al., 2005). In addition, overactivation of CaN has also been reported to increase LDH levels in the rat brain (Wei et al., 2002). Indeed, it is well documented that the neuroprotective effects of FK506 in experimental CNS diseases are associated with reduced neuronal loss, release of antiapoptotic proteins, and promotion of neurite outgrowth (Klettner and Herdegen, 2003; Morita et al., 2008). These findings potentially link CaN to cognitive dysfunction, impairment of cellular oxidant/antioxidant defenses, neurotransmitter deficits, and neuronal cell loss following ICV-STZ administration. FK506 attenuates the learning and memory deficits, cholinergic hypofunction, and oxidative–nitrosative stress induced by ICV-STZ infusion in rats. Conclusion
The observed cognitive enhancement following FK506 treatment in STZ-infused rats may be because of its antioxidant actions and neuromodulatory role over cholinergic neurons, which probably help restore acetylcholine levels. On the basis of the above results, it can be suggested that FK506 may prove to be a useful candidate drug molecule for the management of cognitive disorders such as AD.
Acknowledgements The authors are thankful to Mr Parveen Garg, Chairman, ISF College of Pharmacy, Moga (Punjab) for his praiseworthy inspiration and support for this study.
Conflicts of interest
There are no conflicts of interest.
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FK506 attenuates intracerebroventricular streptozotocininduced neurotoxicity in rats Rimpi B. Arora, Kushal Kumar and Rahul R. Deshmukh Upregulation in calcineurin (CaN) signaling has been implicated in various neurodegenerative disorders. In the present study, we have investigated the effect of FK506 – a CaN inhibitor – on streptozotocin (STZ)induced experimental dementia of the Alzheimer’s type in rats. STZ was administered intracerebroventricularly to induce a cognitive deficit and oxidative stress. Nonimmunosuppressive doses (0.5 and 1 mg/kg postoperatively) of FK506 (tacrolimus) were administered for 21 day in STZ-treated rats. Cognitive functions were assessed using the Morris water maze and passive avoidance tasks. Malondialdehyde and nitrite glutathione levels, as well as acetylcholinesterase activity, were determined to evaluate oxidative stress and cholinergic functions. Lactate dehydrogenase levels were estimated and histological analysis of the dentate gyrus and the CA1 region of the hippocampus was carried out to identify degenerative changes. STZ produced significant deterioration of cognitive functions, oxidative stress, and degenerative changes in the cortical and hippocampal
brain regions. FK506 dose-dependently attenuated STZ-induced cognitive deficits, oxidative stress, and degenerative changes in the cortex and hippocampus. These results suggest a potential role of CaN signaling in degenerative processes, and that inhibition of CaN may be useful in the treatment of neurodegenerative disorders such as Alzheimer’s disease. Behavioural c 2013 Wolters Kluwer Health | Pharmacology 24:580–589 Lippincott Williams & Wilkins.
Introduction
2008), Huntington’s disease (Xifiro et al., 2008; Dudek et al., 2010), and AD (Dineley et al., 2007).
Alzheimer’s disease (AD) is an age-related neurodegenerative disorder (Reed et al., 2009). Accumulating data indicate that disturbances in several aspects of cellular metabolism appear pathologically important in AD. Among these, increased brain insulin resistance (Watson and Craft, 2006; Hoyer and Lannert, 2007; SalkovicPetrisic, 2008) and decreased glucose utilization (Balbo et al., 2002; Steen et al., 2005; De la Torre, 2008) and energy metabolism are observed in the early stages of the disease (De la Monte and Wands, 2005; Hoyer and Lannert, 2007; Salkovic-Petrisic and Hoyer, 2007). Decreased energy metabolism has also been implicated in the activation of numerous signaling pathways and Ca2 + toxicity (Muller et al., 1998). One intracellular signaling element that has recently attracted attention as a potential modulator of both learning and memory function, as well as cell degeneration, is the Ca2 + /calmodulin-dependent protein serine– threonine phosphatase 2B, also known as calcineurin (CaN), which is the most abundant phosphatase in the brain (Mansuy, 2003; Liu et al., 2007) and is activated following Ca2 + influx (Liu, 2009). CaN has been implicated in the pathogenesis of various diseases such as cardiac hypertrophy (Nakamura et al., 2008), congenital heart disease (Groetzner et al., 2005), epilepsy (VazquezLopez et al., 2006), Parkinson’s disease (Wright et al., c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins 0955-8810
Behavioural Pharmacology 2013, 24:580–589 Keywords: cognitive dysfunction, FK506, oxidative stress, rat, sporadic dementia, streptozotocin, tacrolimus Department of Pharmacology, Neuropharmacology Division, ISF College of Pharmacy, Moga, Punjab, India Correspondence to Rahul R. Deshmukh, MPharm, PhD, Department of Pharmacology, Neuropharmacology Division, ISF College of Pharmacy, Moga 142001, Punjab, India E-mail: [email protected] Received 22 January 2013 Accepted as revised 4 July 2013
CaN has been reported to negatively regulate learning and memory (Eto et al., 2008). Inhibition of CaN signaling has been reported to improve associative learning and memory in Tg2876 APP transgenic mice (Dineley et al., 2007) and spatial memory in 3-nitropropionic acidinduced neurotoxic insult (Kumar and Kumar, 2009). CaN is specifically inhibited by certain drugs such as cyclosporine-A and FK506 in the presence of cyclophilin and FK506-binding protein (FKBP-12 and FKBP-52), respectively, resulting in immunosuppressant activity (Van Rossum et al., 2009). In addition, nonimmunosuppressive doses of FK506 have also been demonstrated to provide neuroprotection without affecting inflammatory mechanisms in various models such as experimental stroke (Butcher et al., 1997), spinal-cord injury (Voda et al., 2005), Parkinson’s disease (Wright et al., 2008) and Huntington’s disease (Kumar and Kumar, 2009). Despite these findings, the role of CaN and beneficial effects of FK506 in experimental sporadic dementia of Alzheimer’s type remain elusive. Intracerebroventricular administration of streptozotocin (ICV-STZ) in rats is commonly used to study experimental sporadic dementia of the Alzheimer’s type. ICV-STZ has been reported to produce behavioral, neurochemical, DOI: 10.1097/FBP.0b013e32836546db
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FK506 against STZ-induced neurotoxicity Arora et al. 581
biochemical, and histopathological changes similar to those in an aging brain and is considered to be a suitable animal model of sporadic dementia of the Alzheimer’s type (Prickaerts et al., 1995; Sharma and Gupta, 2001, 2002; Salkovic-Petrisic, 2008; Ishrat et al., 2009). ICV-STZ administration has also been reported to increase the level of cytoplasmic calcium ions (Muller et al., 1998), and the pharmacological blockade of L-type calcium channels (lercanidipine) has been reported to attenuate behavioral and biochemical alterations in ICV-STZ-injected rats (Sonkusare et al., 2005). Dysregulation in Ca2 + homeostasis has been observed in neurodegenerative disorders including AD. Moreover, dysregulation of calcium signaling has been reported to upregulate CaN signaling mechanisms (Foster et al., 2001), which may perhaps contribute to the degenerative changes and subsequent cognitive decline associated with sporadic AD. Thus, in the present study, we have investigated the potential effect of FK506, a CaN inhibitor, on STZ-induced neurotoxicity.
Methods Subjects
The experiments were carried out on male Wistar rats (220–250 g) procured from the Indian Institute of Integrative Medicine, Jammu, India. The animals were kept in polyacrylic cages and maintained under standard housing conditions (room temperature 22±21C and relative humidity of 60–65%), with lights on at 07.00 h and off at 19.00 h, at the ‘Central Animal House’ of ISF College of Pharmacy, Moga, Punjab (India). Food, in the form of dry pellets, and water were freely available. All behavioral experiments were carried out between 10.00 and 16.00 h. The protocol was reviewed and approved by the ‘Institutional Animal Ethics Committee’, and the animal experiments were carried out in accordance with the Indian National Science Academy Guidelines for use and care of animals. Intracerebroventricular infusion of streptozotocin
Rats were anesthetized with ketamine (100 mg/kg, intraperitoneally) and xylazine (5 mg/kg, intraperitoneally). The head was placed in position in the stereotaxic apparatus and a midline saggital incision was made in the scalp. Two holes were drilled through the skull for placement of infusion cannulae into the lateral cerebral ventricles using the following coordinates: 0.8 mm posterior to bregma; 1.5 mm lateral to the saggital suture; 3.6 mm ventral from the surface of the brain (Paxinos and Watson, 1986). After cannula placement, animals were injected with gentamicin (5 mg/kg) and were placed in individual cages. Animals were observed for 1 week, and special care was taken by administering sweetened milk daily during this resting phase for recovery (Fig. 1). After the resting period, STZ was dissolved in citrate buffer (pH 4.4) and slowly infused (1 ml/min), using a Hamilton microsyringe pump (Quintessential stereotaxic injector; Stoelting Co., Wood Dale, Illinois, USA), into each
cerebral ventricle (bilateral ICV) in a volume of 10 ml on days 1 and 3 (Deshmukh et al., 2009). Experimental groups
Animals were divided into five groups, each comprising 10 animals. The treatment schedule and the interval for estimation of various parameters are presented in Fig. 1. Group1, which served as a double-vehicle control, received citrate buffer (pH 4.4) ICV at a volume of 10 ml in each ventricle on days 1 and 3 and 0.5% carboxymethyl cellulose (0.5 ml intraperitoneally, as a vehicle for FK506) for 21 days. The animals in group 2 were treated with 1 mg/kg FK506 postoperatively. Rats in group 3 were infused with ICV-STZ at a dose of 3 mg/kg. Groups 4 and 5 received FK506 at doses of 0.5 and 1 mg/kg, respectively, postoperatively, 1 h after STZ administration; this was started on the first post operative day and continued once daily for a period of 21 days. The nonimmunosuppressive doses of FK506 were selected on the basis of earlier reports in which significant antioxidant and neuroprotective properties were demonstrated at doses ranging from 0.2 to 3 mg/kg (Sakai et al., 1991; Singh et al., 2003a, Gold et al., 2004; Wright et al., 2008). Behavioral assessment Passive avoidance task
On days 14 and 15 after ICV-STZ infusion, the rats were tested for memory retention using a passive avoidance apparatus. The apparatus (Type-7552; Ugo Basile, Comerio, Italy) consisted of a chamber illuminated with a 40-W bulb and a dark chamber, separated by a guillotine door. The floor of the dark chamber consisted of a metal grid with a shock scrambler. During the acquisition trial, the rat was placed in the illuminated chamber. After an initial habituation period of 60 s, the guillotine door was opened and the time taken by the rat to enter the dark chamber was noted. The latency to step into the dark compartment was recorded as the initial trial latency (ITL) or preshock latency. As soon as the rat entered the dark chamber, it was given a mild foot shock of 0.5 mA for 2 s through the grid floor. The rat was allowed to remain in the dark compartment for 5 s and then taken out. After a 24-h interval, a retention trial was performed and retention trial latency (RTL) or postshock latency to step into the dark compartment was noted. The latency was recorded to a maximum of 600 s (Deshmukh et al., 2009). Short latencies indicated poorer retention. Morris water maze
Spatial learning and memory were tested in a Morris water maze (MWM; Morris, 1984), which consisted of a circular water tank (180 cm diameter, 60 cm height) filled with water (25±11C) to a depth of 40 cm. Milk powder was used to render the water opaque. Four equally spaced locations around the edge of the pool (North, South, East, and West) were used as start points, which divided
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Fig. 1
CI
Rest
−1
−7
PAL
ICV-STZ Day1
Day 2
Day 3
Day 14
Day15
MWM
Probe test
Day 17–20
Day 21
Tacrolimus (0.5 and 1 mg/kg, per oral) LA
SAC
Experimental procedure and treatment schedule. CI, cannula implantation; ICV, intracerebroventricular; LA, locomotor activity; MWM, Morris water maze; PAL, passive avoidance latency; SAC, sacrificed; STZ, streptozotocin.
the pool into four quadrants. An escape platform (10 cm in diameter) was placed 2 cm below the water surface in the middle of the north-east quadrant of the pool and kept in the same position throughout the experiment. Before training started, the rats were allowed to swim freely in the pool for 120 s without a platform. Animals underwent a training session consisting of four trials per session (once from each starting point) for 4 days (days 17, 18, 19, and 20), each trial having a ceiling time of 120 s and an intertrial interval of B30 s. After climbing onto the hidden platform, the animals were allowed to remain there for 30 s before commencement of the next trial. If the rat failed to locate the hidden platform within the maximum time of 120 s, it was gently placed onto the platform and allowed to remain there for the same interval of time. Twenty-four hours after the acquisition phase, a probe test (day 21) was conducted by removing the platform. Rats were allowed to swim freely in the pool for 60 s and the percentage of time spent in the target quadrant, which had previously contained the hidden platform, was recorded. The time spent in the target quadrant indicated the degree of memory consolidation that had taken place after learning (Deshmukh et al., 2009). The time taken to locate the hidden platform and the time spent in the target quadrant during the probe trial was measured by an experimenter who was blind to treatment, using a stopwatch. Spontaneous locomotor activity
Each animal was tested for spontaneous locomotor activity on day 21 after the first ICV-STZ infusion. Each animal was observed over a period of 10 min in a square closed arena equipped with infrared light sensitive photocells, using a digital photoactometer (INCO, Ambala, Haryana, India) (Deshmukh et al., 2009). Biochemical parameters Brain homogenate preparation
All biochemical parameters were measured in brain homogenates. On day 22 after the first STZ infusion, animals were killed by decapitation and brains were removed and rinsed with ice-cold isotonic saline. Brain
tissue samples were then homogenized with ice-cold 0.1 mol/l phosphate buffer (pH 7.4) in a volume 10 times the weight of the tissue. The whole brain homogenate was centrifuged at 10 000g for 15 min and aliquots of the supernatant were separated and used for biochemical estimation. Protein estimation
Protein levels in all brain samples were estimated using the method developed by Lowry et al. (1951), with BSA (1 mg/ml) as a standard. Acetylcholinesterase assay
The quantitative measurement of acetylcholinesterase (AChE) activity in the brain was performed according to the method described by Ellman et al. (1961). The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 mol/l sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide, and 0.10 ml of 5,50 -dithiobis(2nitrobenzoic acid) (Ellman reagent). The change in absorbance was measured immediately at 412 nm spectrophotometrically. AChE activity in the supernatant was expressed as nmol/mg protein. Estimation of malondialdehyde
The quantitative measurement of malondialdehyde (MDA), the end product of lipid peroxidation, was performed according to the method developed by Wills (1966). The amount of MDA was measured after its reaction with thiobarbituric acid at 532 nm, using a spectrophotometer (UV-1700; Shimadzu, Columbia, Maryland, USA). The concentration of MDA was determined from a standard curve and expressed as nmol/mg protein. Estimation of reduced glutathione
Reduced glutathione (GSH) levels in the brain were estimated according to the method described by Ellman (1959). One milliliter of supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 41C for 1 h. The samples were centrifuged at 1200g for 15 min. To 1 ml of the supernatant, 2.7 ml of phosphate buffer (0.1 mol/l, pH 8) and 0.2 ml of DTNB were added. The yellow color that developed was measured immediately at 412 nm using a spectrophotometer.
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FK506 against STZ-induced neurotoxicity Arora et al. 583
The concentration of GSH in the supernatant was determined from a standard curve and expressed as mmol/mg protein.
prepared afresh by suspending in 0.5% sodium carboxymethyl cellulose. All other chemicals used in the study were of analytical grade. Solutions of the drug and chemicals were freshly prepared before use.
Estimation of nitrite
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined by a colorimetric assay using the Greiss reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid], as described by Green et al. (1982). Equal volumes of the supernatant and Greiss reagent were mixed, the mixture was incubated for 10 min at room temperature in the dark, and the absorbance was determined at 540 nm spectrophotometrically. The concentration of nitrite in the supernatant was determined from the sodium nitrite standard curve and expressed as mmol/mg protein.
Statistical analysis
Assessment of histological changes
On day 14 after the first ICV-STZ injection, the mean initial latency in the acquisition trial remained unchanged among all the groups [F(4,49) = 2.21, NS]. However, the mean retention latency on day 15 was significantly decreased (P < 0.001) in the ICV-STZ control group compared with vehicle-treated animals (Fig. 2). The ICV-STZ-induced decrease in the mean retention latency was significantly increased on FK506 treatment [F(4,49) = 174.3, P < 0.001], indicating protection against the effect of STZ treatment. The maximum improvement in retention latency by FK506 treatment on day 15 was observed at a dose of 1 mg/kg (P < 0.001). Moreover, control rats treated with FK506 (1 mg/kg) alone did not show any significant difference in retention latency as compared with vehicle-treated rats (P > 0.05).
The brains were rapidly removed and fixed by immersion in 10% formalin. Subsequently they were embedded in paraffin wax, cut into 5-mm-thick sections, and stained with cresyl violet acetate (Nissl stain; Shoham et al., 2003). Hippocampal brain sections were examined under bright field illumination (AHBT-51, Olympus VanoxAHBT; Olympus America, Melville, New York, USA). Drugs
STZ, acetylthiocholine iodide, and DTNB were purchased from Sigma-Aldrich (St Louis, Missouri, USA). FK506 was provided as an ex-gratia sample by M/S Panacea Biotec Ltd. (Mohali, Punjab, India). STZ was diluted in citrate buffer (pH 4.4) and FK506 was always
The results are expressed as mean±SD. The behavioral (for individual time points) and biochemical values were analyzed by one-way analysis of variance, followed by Tukey’s post-hoc test for multiple comparisons, using GraphPad Prism statistical software (version 5.0; GraphPad Prism software Inc., La Jolla, California, USA). A P-value of less than 0.05 was set to be statistically significant.
Results Effect of FK506 on memory performance in the passive avoidance task in ICV-STZ-treated rats
Fig. 2
Transfer latency (s)
600
Day-14 aquisition trial
Day-15 retention trial
∗#
400 ∗
200 @
g)
g)
m (1 FK
(0
.5
m
ST Z FK
e Pe
rs
cl e FK
m
hi Ve
g)
g) (1 FK
(0
.5
m
ST Z FK
e Pe rs
FK
Ve hi
cl
e
0
Effect of FK506 on performance of ICV-STZ-treated rats in the passive avoidance task. Values are expressed as mean±SD; @P < 0.05 versus vehicle; *P < 0.05 versus STZ; #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
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584 Behavioural Pharmacology 2013, Vol 24 No 7
Effect of FK506 on memory performance in the MWM task in ICV-STZ-treated rats
Effect of FK506 on brain GSH levels in ICV-STZ-treated rats
On day 17 of the acquisition trial, experimental animals from all the groups took a similar amount of time to locate the hidden platform in the MWM task [F(4,49) = 1.109, P > 0.05], but on days 18, 19, and 20 of the acquisition phase, STZ-injected rats showed a significant impairment in learning relative to the vehicle control group [F(4,49) = 59.07, 34.12, and 71.83, respectively, P < 0.001] (Fig. 3a). Moreover, during the probe trial [F(4,49) = 39.64, P < 0.01], STZ-treated rats also failed to remember the precise location of the platform, spending significantly less time in the target quadrant as compared with vehicletreated animals (P < 0.001; Fig. 3b). Chronic administration of FK506 in STZ-treated rats significantly and dosedependently attenuated STZ-induced acquisition and retention deficits in the MWM task. In the probe trial, FK506-treated rats spent more time in the target quadrant, as compared with STZ-treated control rats, indicating improved consolidation of memory (P < 0.05).
STZ administration in rats produced a significant depletion in GSH levels in the brain [F(4,49) = 83.90, P < 0.001] (Table 1) as compared with vehicle treatment (P < 0.001). Chronic administration of FK506 significantly restored GSH levels in STZ-treated rats (P < 0.01) without affecting GSH levels in control rats.
Effect of FK506 on spontaneous locomotor activity in ICV-STZ-treated rats
Effect of FK506 on the neuronal morphology of the dentate gyrus and the CA1 region of the hippocampus in ICV-STZ-treated rats
Spontaneous locomotor activity on day 21 did not differ significantly among groups [F(4,49) = 0.41, NS] (Fig. 4). Effect of FK506 on brain AChE activity in ICV-STZtreated rats
Ventricular administration of STZ produced a significant increase in brain AChE activity relative to vehicle controls [F(4,34) = 63.28, P < 0.001] (Fig. 5). Chronic administration of FK506 (0.5 and 1 mg/kg) dose-dependently attenuated the STZ-induced increase in AChE activity (P < 0.01). However, FK506 did not affect AChE activity in control rats (P > 0.05). Effect of FK506 on MDA levels in ICV-STZ-treated rats
Administration of STZ in rats produced a pronounced elevation in MDA levels, as compared with vehicle treatment [F(4,34) = 92.38, P < 0.001] (Table 1). Chronic administration of FK506 dose-dependently attenuated the STZ-induced elevation in MDA levels (P < 0.001). However, FK506 treatment did not produce any significant effect in normal animals when compared with vehicle controls (P > 0.05). Effect of FK506 on brain nitrite level in ICV-STZ-treated rats
STZ-treated rats showed a significant increase in brain nitrite levels as compared with vehicle control-treated animals [F(4,34) = 98.44, P < 0.001] (Table 1). The STZinduced increase in nitrite levels was significantly and dose-dependently attenuated by FK506 (0.5 and 1 mg/kg, P < 0.01), whereas FK506 administration did not produce any significant effect (P > 0.05) in control animals as compared with vehicle-treated animals.
Effect of FK506 on brain lactate dehydrogenase levels in ICV-STZ-treated rats
STZ produced a significant elevation in brain lactate dehydrogenase (LDH) levels [F(4,34) = 59.67, P < 0.001] (Fig. 6) as compared with vehicle treatment, indicating cellular damage. FK506 (0.5 and 1 mg/kg) dose-dependently attenuated the STZ-induced elevation in LDH levels (P < 0.01). However, FK506 treatment did not alter LDH levels in the brains of normal rats as compared with vehicle treatment (P > 0.05).
Cresyl violet-stained hippocampal dentate gyrus and CA1 sections of vehicle-treated animals showed healthy neurons (Fig. 7a, b), which were robust in shape, had a pale and spherical or slightly oval nucleus, and had a single large nucleolus with a clear, visible cytoplasm. Brain sections from rats treated with FK506 (1 mg/kg, postoperatively) also showed healthy neurons in the dentate gyrus and CA1 region (Fig. 7c, d). Similar morphology and neuronal density in the dentate gyrus and the hippocampal CA1 region were observed in the vehicle-treated and FK506-treated groups (Fig. 7a–d). Neuronal degeneration may present a continuum of neurodegenerative morphologies. In this study, brain sections from ICV-STZ-treated rats showed pyknotic neurons that were darkly stained with no nucleus or visible nucleolus, and a few of the cells were shrunken and sickle shaped. Photomicrographs of the dentate gyrus and CA1 region of the hippocampus of ICV-STZ-treated groups showed a marked decrease in cell bodies (Fig. 7e, f). Other neurons showed a smaller nucleus with condensed chromatin and a swollen cytoplasm. Further, STZ administration markedly decreased neuronal density and increased the number of pyknotic neurons. Chronic treatment with FK506 (0.5 mg, Fig. 7g, h; 1 mg, Fig. 7i, j) attenuated STZ-induced cell loss and pyknosis; however, some degenerating cells with changes in morphology were observed in the FK506 treatment groups. Sections from rats treated with the higher dose of FK506 showed rounded and open nuclei with marked protection from STZ-induced neurodegenerative morphologies (Fig. 7i, j). There was marked improvement in neuronal density and reduction in pyknotic neurons following chronic FK506 treatment in STZ-treated rats, indicating a neuroprotective potential, in line with behavioral and biochemical effects of FK506.
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FK506 against STZ-induced neurotoxicity Arora et al. 585
Fig. 3
(a)
120
Vehicle
FK per se
STZ
FK (0.5 mg)
FK (1 mg)
Mean escape latency (s)
100 @
80
∗
@
@
∗# 60
∗ ∗#
∗
40
∗# 20 0 Day 17
Time spent in target quadrant (%)
(b)
Day 18
Day 19
Day 20
100
80 ∗# 60
40
∗ @
20
0 Vehicle
FK per se
STZ
FK (0.5 mg)
FK (1 mg)
Effect of FK506 on performance of ICV-STZ-treated rats in the Morris water maze. (a) Escape latency and (b) time spent in the target quadrant. Values are expressed as mean±SD; @P < 0.05 versus vehicle, *P < 0.05 versus STZ, #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin. Effect of tacrolimus on time spent in target quadrant in ICV-STZ-treated rats is expressed as mean±SD. @ P < 0.05 versus Vehicle, *P < 0.05 versus STZ, #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
Discussion The present study demonstrated a neuroprotective role of FK506, a specific CaN inhibitor, against STZ-induced behavioral, biochemical, and histological toxicity in rats. In line with earlier reports, ICV-STZ was found to produce significant impairment in spatial cognitive functions and increase oxidative–nitrosative stress (Sharma and Gupta, 2001; Sonkusare et al., 2005; Ishrat et al., 2006; Salkovic-Petrisic and Hoyer, 2007; SalkovicPetrisic, 2008). FK506 dose-dependently (0.5 and 1 mg/kg postoperatively) protected against the effect of STZ treatment in the MWM and passive avoidance task (Fig. 2 and Fig. 3a, b) without affecting locomotor activity (Fig. 4). Changes in locomotor activity have been suggested to modulate learning and memory in the passive avoidance and MWM paradigms (Sharma and
Gupta, 2003; Deshmukh et al., 2009). However, no significant difference in spontaneous locomotor activity was observed in any of the experimental groups in the present study. The cholinergic system plays an important role in memory formation and retrieval (Olton, 1990; Fibiger, 1991; Fibiger et al., 1991; Blokland, 1995). In the present study, STZ-induced cognitive deficit was also associated with an increase in cholinesterase activity (Fig. 5), which is in accordance with the findings of earlier studies (Sonkusare et al., 2005; Ishrat et al., 2006; Deshmukh et al., 2009). In the present study, FK506 dose-dependently protected against the effect of STZ treatment in the MWM and passive avoidance task and restored cholinesterase activity without modifying basal memory and cholinesterase
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586 Behavioural Pharmacology 2013, Vol 24 No 7
Fig. 4
Fig. 6
500 LDH levels (IU/mg protein)
Activity counts/10 min
300
200
100
0
@
400 300
∗ ∗#
200 100 0
Vehicle
FK per se
STZ
FK 0.5 mg
FK 1 mg
Effect of FK506 on spontaneous locomotor activity in ICV-STZ-treated rats. Values are expressed as mean±SD. FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
Vehicle
FK per se
STZ
FK 0.5 mg
FK 1 mg
Effect of FK506 on brain lactate dehydrogenase in ICV-STZ-treated rats. Values are expressed as mean±SD; @P < 0.05 versus vehicle; *P < 0.05 versus STZ; #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
Fig. 5
Acetylcholinesterase activity (nmol/mg protein)
500
@
400 ∗ 300
∗#
200 100 0 Vehicle
FK per se
STZ
FK (0.5 mg) FK (1 mg)
Effect of FK506 on brain acetylcholinesterase activity in ICV-STZtreated rats. Values are expressed as mean±SD; @P < 0.05 versus vehicle; *P < 0.05 versus STZ; #P < 0.05 versus FK506 (0.5 mg/kg). FK, FK506; ICV, Intracerebroventricular; LDH, lactate dehydrogenase; STZ, streptozotocin.
Table 1
Effect of FK506 on biochemical parameters Biochemical parameters
Groups Vehicle FK per se STZ FK (0.5 mg) FK (1 mg)
MDA (nmol/mg protein)
GSH (mmol/mg protein)
Nitrite (mmol/mg protein)
168.6±16.57 163.2±27.44 411.2±40.71@ 278.4±27.75* 213.6±23.32*#
1.84±0.23 1.95±0.28 0.18±0.08@ 0.91±0.18* 1.31±0.21*#
0.43±0.25 0.42±0.18 3.47±0.54@ 2.20±0.33* 1.55±0.28*#
Values are expressed as mean±SD. FK, FK506; GSH, reduced glutathione; MDA, malondialdehyde; STZ, streptozotocin. @ P < 0.05 versus vehicle. *P < 0.05 versus STZ. # P < 0.05 versus FK506 (0.5 mg/kg).
activity. CaN is a Ca2 + /calmodulin-dependent protein serine–threonine phosphatase 2B and the most abundant phosphatase in the central nervous system (CNS)
(Mansuy, 2003; Liu et al., 2007). Impairment of insulin signaling and glucose and energy metabolism following administration of STZ into the CNS has been suggested to cause Ca2 + overactivation (De la Monte and Wands, 2005; Sonkusare et al., 2005), which may lead to further imbalance between kinases and phosphatases (SalkovicPetrisic and Hoyer, 2007; Salkovic-Petrisic, 2008). Moreover, it has been reported that CaN is the first among the postsynaptic phosphatases to be activated in response to Ca2 + influx (Liu, 2009). Indeed, ICV-STZ administration has been reported to increase intracellular Ca2 + concentration (Muller et al., 1998), and blockade of the L-type calcium channel by lercanidipine has been reported to improve cholinergic function and cognitive behavior in STZ-treated rats (Sonkusare et al., 2005). In the present study, STZinduced cognitive deficits and the increase in AChE levels were significantly attenuated by FK506. It can be speculated that the increase in intracellular Ca2 + concentration following STZ administration may subsequently lead to CaN overactivation and contributes to cognitive deficit. Indeed, overactivation of CaN has been reported to negatively regulate learning and memory (Mansuy, 2003; Baumgartel et al., 2008). In addition, acute inhibition of CaN by FK506 has been reported to restore associative learning and memory in Tg2576 APP mice (Dineley et al., 2007; Taglialatela et al., 2009). Oxidative stress has been implicated in the pathogenesis of AD in humans (Pratico, 2008; Mangialasche et al., 2009). Oxyradical-induced damage to macromolecules (lipids, proteins, and nucleic acids, etc.) is considered an important factor in the acceleration of aging and agerelated neurodegenerative disorders such as AD (Liu et al., 2001; Wickens, 2001). An STZ-induced impairment in cerebral energy metabolism and mitochondrial disturbances may be responsible for increased intracellular Ca2 + influx and generation of reactive oxygen species,
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FK506 against STZ-induced neurotoxicity Arora et al. 587
Fig. 7
(a, c, e, g, i) Cresyl violet-stained sections of the dentate gyrus and (b, d, f, h, j) the CA1 region of the hippocampus following treatment with (a, b) vehicle, (c, d) FK506 (1 mg/kg), (e, f) ICV-STZ, and chronic treatment with (g, h) 0.5 mg FK506 and (i, j) 1 mg FK506 following ICV-STZ treatment. (d, f, h, j) Pictures taken at a magnification of 400. FK, FK506; ICV, intracerebroventricular; STZ, streptozotocin.
leading to oxidative stress (Muller et al., 1998; Shoham et al., 2007; Deshmukh et al., 2009). Further, ICV administration of STZ has been reported to cause Ca2 + -dependent production of NO, a precursor for free radicals, which reacts with superoxide anions to form peroxynitrite, causing nitrosative stress (Shoham et al., 2007; Kumar and Kumar, 2009). Consistent with previous studies, in the present study, ICV-STZ treatment significantly increased MDA and nitrite levels and decreased the levels of endogenous antioxidant enzymes, indicating increased oxidative–nitrosative stress (Deshmukh et al., 2009). Chronic treatment of STZ-treated rats with FK506 dose-dependently attenuated the STZ-induced increase in oxidative–nitrosative stress. These results suggest that FK506 may have direct antioxidant potential
or may decrease free radical generation by inhibiting CaN activity. It has been reported that oxidative stress regulates Ca2 + dependent serine–threonine phosphatase, that is, CaN (Manik et al., 2003). In addition, mitochondrial dysfunction and oxidative stress have been observed in CaN transgenic mice, indicating that the upregulation of CaN signaling may lead to oxidative burden (Sayen et al., 2003). In addition, CaN has been reported to dephosphorylate nitric oxide synthase, resulting in an increase in NO production (Knirsch et al., 2001). Indeed, FK506, by CaN inhibition, has been reported to negatively regulate Ca2 + -channel activity and inhibit free radical generation and nitric oxide synthase activity
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588 Behavioural Pharmacology 2013, Vol 24 No 7
(Victor et al., 1995; Phillis et al., 2002; Sasaki et al., 2004). In addition, FK506 has also been reported to possess direct free radical scavenging properties and to improve antioxidant defense mechanisms (Maziere et al., 2005). Therefore, FK506 may produce its antioxidant effect directly through free radical scavenging or indirectly through CaN inhibition. In the brain, STZ has been reported to cause neurodegeneration accompanied by an increase in LDH activity (Hoyer and Lannert, 2007). Consistent with these findings, in the present study, ICV-STZ-treated animals showed a marked increase in LDH levels, indicating cell damage. However, chronic administration of FK506 significantly attenuated the STZ-induced elevation in LDH levels. In line with the biochemical alterations, histological sections of the dentate gyrus and the CA1 region of the hippocampus of STZ-treated rats showed decreased neuronal density and necrotic cell death, as evidenced by pyknotic nuclear changes. Chronic administration of FK506 dose-dependently attenuated STZ-induced neurotoxicity and was able to restore neuronal density and a normal morphological pattern (Fig. 7), suggesting a neuroprotective potential of FK506. Ca2 + mobilization following stimulation of N-methyl-Daspartate receptors by glutamate has been implicated in cell death in the CNS. The Ca2 + sensitivity of nitric oxide synthase makes the Ca2 + -dependent phosphatase, CaN, an obvious candidate that affects neuronal cell death (Dawson et al., 1993), and therefore, CaN may be a material factor in Ca2 + -activated neuronal cell death (Shibasaki and Mckeon, 1995; Norris et al., 2005). In addition, overactivation of CaN has also been reported to increase LDH levels in the rat brain (Wei et al., 2002). Indeed, it is well documented that the neuroprotective effects of FK506 in experimental CNS diseases are associated with reduced neuronal loss, release of antiapoptotic proteins, and promotion of neurite outgrowth (Klettner and Herdegen, 2003; Morita et al., 2008). These findings potentially link CaN to cognitive dysfunction, impairment of cellular oxidant/antioxidant defenses, neurotransmitter deficits, and neuronal cell loss following ICV-STZ administration. FK506 attenuates the learning and memory deficits, cholinergic hypofunction, and oxidative–nitrosative stress induced by ICV-STZ infusion in rats. Conclusion
The observed cognitive enhancement following FK506 treatment in STZ-infused rats may be because of its antioxidant actions and neuromodulatory role over cholinergic neurons, which probably help restore acetylcholine levels. On the basis of the above results, it can be suggested that FK506 may prove to be a useful candidate drug molecule for the management of cognitive disorders such as AD.
Acknowledgements The authors are thankful to Mr Parveen Garg, Chairman, ISF College of Pharmacy, Moga (Punjab) for his praiseworthy inspiration and support for this study.
Conflicts of interest
There are no conflicts of interest.
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FK506 against STZ-induced neurotoxicity Arora et al. 589
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