Neurochem Res (2011) 36:1062–1072 DOI 10.1007/s11064-011-0450-1

ORIGINAL PAPER

Oxidative Stress in a Model of Toxic Demyelination in Rat Brain: The Effect of Piracetam and Vinpocetine Omar M. E. Abdel-Salam • Yasser A. Khadrawy Neveen A. Salem • Amany A. Sleem

•

Accepted: 15 March 2011 / Published online: 30 March 2011 Ó Springer Science+Business Media, LLC 2011

Abstract We studied the role of oxidative stress and the effect of vinpocetine (1.5, 3 or 6 mg/kg) and piracetam (150 or 300 mg/kg) in acute demyelination of the rat brain following intracerebral injection of ethidium bromide (10 ll of 0.1%). Results: ethidium bromide caused (1) increased malondialdehyde (MDA) in cortex, hippocampus and striatum; (2) decreased total antioxidant capacity (TAC) in cortex, hippocampus and striatum; (3) decreased reduced glutathione (GSH) in cortex and hippocampus (4); increased serum nitric oxide and (5) increased striatal (but not cortical or hippocampal) acetylcholinesterase (AChE) activity. MDA decreased in striatum and cortex by the lower doses of vinpocetine or piracetam but increased in cortex and hippocampus and in cortex, hypothalamus and striatum by the higher dose of vinpocetine or piracetam, respectively along with decreased TAC. GSH increased by the higher dose of piracetam and by vinpocetine which also decreased serum nitric oxide. Vinpocetine and piracetam displayed variable effects on regional AChE activity. Keywords Toxic demyelination  Ethidium bromide  Vinpocetine  Piracetam  Rat brain

O. M. E. Abdel-Salam (&)  N. A. Salem Department of Toxicology and Narcotics, National Research Centre, Tahrir St., Dokki, Cairo, Egypt e-mail: [email protected] O. M. E. Abdel-Salam  A. A. Sleem Department of Pharmacology, National Research Centre, Cairo, Egypt Y. A. Khadrawy Department of Physiology, National Research Centre, Cairo, Egypt

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Introduction Demyelinating diseases are a group of disorders characterized by loss of white matter in discrete areas of the central nervous system. Multiple sclerosis is by far the most common inflammatory autoimmune demyelinating disease of the central nervous system and a leading cause of neurological disability in young adults in which the appearance of focal neurological deficits, disseminated in time and place is due to an immunological attack on the myelin sheath in the central nervous system. The hallmark of the disease is the presence of discrete areas of focal inflammation, demyelination and axonal loss with scarring. This is followed by nerve conduction deficits which is likely to be the most significant factor in causing the neurological disability. Treatment still presents a challenge [1–3]. Oxidative stress defined as a breach in the balance between the production of reactive oxygen species and endogenous antioxidant mechanisms, is a consequence of aerobic metabolism, where toxic reactive intermediates results from the partial reduction of molecular oxygen in the mitochondrial transport chain [4]. Free radicals arise in the brain also from activated microglia, dopamine metabolism, auto-oxidation of dopamine into reactive dopamine quinines and superoxide radical. An attack on lipids, proteins and nucleic acids and their oxidative modification is followed by structural and functional perturbations. In the brain, the high content of polyunsaturated fatty acids and the high utilization of oxygen account for the susceptibility to free radical damage. Oxidative stress is likely to have an important role in aging [5] and in the development of a number of neurodegenerative disorders, where a shared reduction in Fe, Zn and anti-oxidant capacity and an increase in oxidative status has been observed in the serum of patients with Alzheimer’s disease, Parkinson’s disease as well as multiple sclerosis [6].

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It has also been suggested that the action of methylprednisolone in achieving remission in multiple sclerosis patients involves antioxidant mechanism through increasing serum levels of uric acid, a strong peroxynitrite scavenger [7]. Piracetam (2-oxo-1-pyrrolidine-acetamide) is a cyclic derivative of gamma-aminobutyric acid that has been shown to preserve memory from disruption under different experimental conditions [8]. The drug is being widely used in man to treat cognitive impairment in a number of clinical situations. When given to stroke patients with aphasia, the drug improved recovery of various language functions and increased task-related blood flow activation [9]. The drug also improved cognitive function in the elderly [10] and after coronary artery bypass [11] and improved degenerative cerebellar ataxia [12]. Piracetam is largely thought to benefit the ischaemic brain through increasing blood flow, affecting membrane fluidity [13] and glucose transport into the cell [14]. Piracetam reversed hippocampal membrane alterations in Alzheimer’s disease [15] and inhibited the lipid-destabilising effect of the amyloid peptide Abeta C-terminal fragment [16]. Vinpocetine (vinpocetine-ethyl apovincaminate), is a synthetic derivative of the alkaloid vincamine, an extract of periwinkle (Vinca minor) that displayed memory protective properties [17] and improved cognitive function in patients with cerebrovascular disease [18]. It has been shown to increase cerebral blood flow and regional cerebral glucose uptake. The drug is a phosphodiesterase inhibitor, selective for PDE1 [19] and a blocker of voltage-gated Na? channels [20], which is likely to be especially relevant to the neuroprotective effect of vinpocetine. An antioxidant activity of the drug could also contribute to the neuroprotection [21, 22]. Thus, although these two drugs designated as nootropics or memory enhancing agents, a term introduced by Griuga [23] are primarily used in the treatment of memory disorders in man especially that occurring as a apart of normal aging or due to some pathological processes e.g., Alzheimer’s disease, there are data to suggest a neuroprotective [24, 25] and possible antioxidant properties [21, 22, 25, 26] for both of them. Whether these drugs would have a place in treatment of brain demyelination is not known. Therefore it looked pertinent to test whether these drugs would have a place in treatment of brain demyelination. A commonly used model to evoke demyelination involves the local injection of the DNA chelating agent ethidium bromide into the brain or spinal cord of rat or mice. This DNA chelating agent evokes transient demyelination which can be used to study the pathogenetic mechanisms and the possible therapeutic interventions [27, 28]. The aims of the present study were therefore to: (1) assess the oxidative status in the brain tissue of rats subjected to an injection of ethidium bromide; (2) test for the possible

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modulatory effect for vinpocetine and piracetam; two commonly used neuroprotective and memory enhancing drugs.

Experimental Procedure Animals Thirty five adult male Sprague Dawly rats weighing (130 ± 10 g) were used in this study. The animals were obtained from the Animal House Colony of the National Research Centre (Cairo, Egypt). They were housed in stainless steel wire meshed suspended rodent cages under environmentally controlled conditions. The ambient temperature was 25 ± 2°C and the light/dark cycle was 12/12 h. The animals had free access to water and standard rodent chow diet. All animals received human care in compliance with guidelines of the Ethical Committee of National Research Centre, Egypt Centre and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). Equal groups of 5 rats each were used in all experiments. Drugs and Chemicals Ethidium bromide (Sigma, USA), vinpocetine (Vinporal, Amrya. Pharm. Ind., Cairo, ARE), piracetam (Nootropil, Chemical Industries Development;CID, Cairo, ARE) were used. All drugs were dissolved in isotonic (0.9% NaCl) saline solution immediately before use. Surgical Procedures Rats were anaesthetized with sodium pentobarbital (40 mg/ kg, i.p.). and after shaving the hair from the fronto-occipital area antisepsis was performed with 2% iodine solution. A hole of 0.5 cm was made using orthodontic roof motor and number 2 drill to the right of the bregma until the dura matter was exposed. With the use of a Hamilton syringe fitted with a 30-gauge needle the solution of ethidium bromide (10 ll of 1%) was injected in the cisterna pontis (basal), an enlargement of the subarachnoid space on the ventral surface of the pons. A group of rats (n = 5) has undergone to the same surgical procedure but injected with saline (0.9%) and served as negative control. The dura matter was left open and the skin together with remainder of the subcutaneous tissue was sutured with a nylon thread 4.0. Experimental Design Starting on the 2nd day of ethidium bromide injection rats were randomly assigned into 6 groups (n = 5 each) and

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received the following treatments intraperitoneally for 7 days. Negative control and positive control animals were injected daily with saline. The rest of the ethedium bromide-injected rats were treated daily with either vinpocetine (1.5, 3 or 6 mg/kg) (groups 2–4) or piracetam (150 or 300 mg/kg) (groups 5, 6). Blood samples were collected from the retro-orbital vein plexus, under ether anaesthesia in clean test tubes and sera were separated using cooling centrifuge. Then animals were killed by decapitation in deep ether anesthesia. Brains were then removed, one portion was washed with ice-cold saline solution (0.9% NaCl), and sectioned into cortex, hippocampus and striatum, weighed and stored at -80°C for further determination of biochemical parameters. The brain was homogenised with 0.1 M phosphate buffer saline at pH 7.4, to give a final concentration of 10% w/v for the biochemical assays. Biochemical Studies Determination of Brain Lipid Peroxidation Lipid peroxidation was assayed by measuring the level of malondialdehyde (MDA) in the brain tissues. Malondialdehyde was determined by measuring thiobarbituric reactive species using the method of Ruiz-Larrea et al. [29] in which the thiobarbituric acid reactive substances react with thiobarbituric acid to produce a red colored complex having peak absorbance at 532 nm.

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measured at 412 nm and the GSH concentration was calculated by comparison with a standard curve. Determination of Brain Glucose Glucose was measured in supernatants of brain homogenates by a standard glucose oxidase method according to Trinder [32]. Glucose in the presence of glucose oxidase is converted to peroxide and gluconic acid. The produced hydrogen peroxide reacts with phenol and 4-amino-antipyrine in the presence of peroxidase to yield a colored quinonemine, which is measured spectrophotometrically. Determination of Brain Acetylcholinesterase Activity The procedure used for the determination of acetylcholinesterase activity in the hippocampus and cortex was a modification of the method of Ellman et al. [33] as described by Gorun et al. [34]. The principle of the method is the measurement of the thiocholine produced as acetylthiocholine is hydrolyzed. The colour was read immediately at 412 nm. Determination of Serum Nitric Oxide Level Serum nitric oxide measured as nitrite was determined by using Griess reagent, according to the method of Moshage et al. [35], where nitrite, stable end product of nitric oxide radical, is mostly used as indicator for the production of nitric oxide.

Determination of Brain Total Antioxidant Capacity Statistical Analysis Antioxidant capacity in brain tissue is performed by the reaction of antioxidants in the sample with a defined amount of exogenously provided hydrogen peroxide. The antioxidants eliminate a certain amount of peroxide. The residual peroxide is determined colorimetrically by an enzymatic reaction which involves the conversion of 3,5, dichloro-2-hydroxy benzensulphate to a colored product [30].

Data are expressed as mean ± SE. Data were analyzed by one-way analysis of variance, followed by Duncan’s multiple range test for post hoc comparison of group means. Effects with a probability of P \ 0.05 were considered to be significant.

Results Determination of Brain Reduced Glutathione Content Lipid Peroxidation Reduced glutathione (GSH) was determined in brain tissue by Ellman’s method [31]. The procedure is based on the reduction of Ellman’s reagent by –SH groups of GSH to form 2-nitro-s-mercaptobenzoic acid, the nitromercaptobenzoic acid anion has an intense yellow color which can be determined spectrophotometrically. A mixture was directly prepared in a cuvette: 2.25 ml of 0.1 M K-phosphate buffer, pH 8.0; 0.2 ml of the sample; 25 ll of Ellman’s reagent (10 mM 5,50 -dithio-bis-2-nitrobenzoic acid in methanol). After 1 min the assay absorbance was

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Following injection of ethidium bromide, there was a significant increase in lipid peroxidation in the cortex by 56.9% (30.1 ± 0.24 vs. 19.18 ± 0.21 nmol/g, P \ 0.05), hippocampus by 19.6% (29.66 ± 0.14 vs. 24.79 ± 0.21, nmol/g, P \ 0.05) and in striatum by 22.8% (22.34 ± 0.20 vs. 18.18 ± 0.13 nmol/g, P \ 0.05) compared with the salinetreated group. Vinpocetine given at 1.5 mg/kg to ethidium bromide-treated rats decreased striatal MDA levels by 19.8% (17.91 ± 0.12 vs. 18.18 ± 0.13 nmol/g, P \ 0.05).

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Vinpocetine given at 6 mg/kg increased MDA in cortex and hippocampus by 17 and 17.5%, respectively, compared with ethidium bromide control value. With piracetam at 150 mg/ kg there was a 16.2 and 11.6% decrease of MDA in cortex compared to ethidium bromide control value (25.2 ± 0.11 vs. 19.18 ± 0.21 and 19.74 ± 0.18 vs. 23.34 ± 0.2 nmol/ g, respectively, P \ 0.05). The higher dose of the drug increased MDA in cortex, hippocampus and striatum by 22.8, 35.1 and 24.5%, respectively (P \ 0.05 for all). Values of MDA in nmol/g were: cortex, 36.96 ± 0.23; hippocampus, 40.07 ± 0.20; striatum, 27.82 ± 0.23 after ethidium bromide ?300 mg/kg piracetam versus ethidium bromide (positive control) values of: cortex, 19.18 ± 0.21; hippocampus, 24.79 ± 0.21; striatum, 18.18 ± 0.13. In summary, MDA is increased in cortex, hippocampus and striatum after ethidium bromide. MDA is decreased in striatum by the low dose of vinpocetine and in cortex and striatum by the low dose of piracetam. MDA is increased in cortex and hippocampus by the high dose of vinpocetine and in all brain areas by the high dose of piracetam. Thus lower doses of either vinpocetine or piracetam decreased, while higher doses increased lipid peroxidation (Fig. 1).

tissue, P \ 0.05) versus saline-treated control. No significant change in TAC was observed in rats treated with 1.5 mg/kg vinpocetine. TAC showed further reduction in the hippocampus and striatum by the highest dose of vinpocetine (19.7 and 47.4% decrease vs. ethidium bromide control value; 6.2 ± 0.4 vs. 7.72 ± 0.4 lmol/g tissue, P \ 0.05 and 6.1 ± 0.4 vs. 11.6 ± 1.3 lmol/g tissue, P \ 0.05, respectively). It showed non-significant increased in the hippocampus (by 21.8%, P [ 0.05) after 3 mg/kg of vinpocetine and in the cortex (by 23.1%, P [ 0.05) after 6 mg/kg of vinpocetine, respectively. TAC decreased in all brain areas examined by either dose of piracetam. It decreased by 53.7, 23.6 and 50% in cortex, hippocampus and striatum, respectively, after 150 mg/kg piracetam and by 17.7, 14.5 and 20.7% after 300 mg/kg piracetam, indicating a further decrease in TAC after the administration of piracetam. The above results indicated that TAC is decreased by ethidium bromide in cortex, hippocampus and striatum. TAC is decreased in the hippocampus and striatum by the highest dose of vinpocetine and decreased in all brain areas by either dose of piracetam (Fig. 2). Reduced Glutathione

Total Antioxidant Capacity Total antioxidant capacity (TAC) was decreased by ethidium bromide in the cortex by 30% (5.71 ± 0.9 vs. 8.16 ± 0.9 lmol/g tissue, P \ 0.05), in hippocampus by 32.3% (7.72 ± 0.4 vs. 11.4 ± 0.7 lmol/g tissue, P \ 0.05) and in striatum by 30.9% (11.6 ± 1.3 vs. 16.8 ± 1.7 lmol/g

The administration of ethidium bromide resulted in a significant decrease of reduced glutathione (GSH) in both the cortex and hippocampus by 31.4% (P \ 0.05) and 36.1% (P \ 0.05), respectively compared with the saline-treated group (6.45 ± 0.42 vs. 9.41 ± 0.66 lg/g and 6.72 ± 0.41 vs. 10.52 ± 0.84 lg/g, respectively). A non-significant

Hippocampus

Malondialdehyde (nmol/g tissue)

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Cortex

35.1%

40 19.6%

35

*

22.8%

*

*

17.5%

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Striatum 24.5%

30

*

-16.3%

25

*

20

-19.8%

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Saline Ethidium control + Vinpocetine 1.5 mg/kg + Vinpocetine 3 mg/kg + Vinpocetine 6 mg/kg + Piracetam 150 mg/kg + Piracetam 300 mg/kg

-11.6%

*

15 10 5 0

Fig. 1 The effect of treatment with vinpocetine (1.5, 3 or 6 mg/kg), piracetam (150 or 300 mg/kg) on the brain tissue (cortex, hippocampus and striatum) levels of malondialdehyde (MDA: nmol/g tissue) in rats given an intracerebral injection of the demyelinating agent ethidium bromide (10 ll of 0.1%). MDA is increased in cortex, hippocampus and striatum after ethidium bromide. MDA is decreased in striatum by the low dose of vinpocetine and in cortex and striatum by the low dose of piracetam. MDA is increased in cortex and

hippocampus by the high dose of vinpocetine and in all brain areas by the high dose of piracetam. Data are expressed as the mean ± SE. n = 5. The percent change from ethidium bromide control group is shown on top of bars. Data were analyzed by one-way analysis of variance, followed by a Tukey’s multiple range test for post hoc comparison of group means. Effects with a probability of P \ 0.05 were considered to be significant. Asterisks indicate significant change from ethidium bromide control group

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Total antioxidant capacity (µmol/ g tissue)

Striatum 18

15

Hippocampus 12

21.8%

Cortex 9

6

-20.7%

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*

23.1%

-23.6% -14.5%

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-19.7% -17.7%

Saline Ethidium control + Vinpocetine 1.5 mg/kg + Vinpocetine 3 mg/kg + Vinpocetine 6 mg/kg + Piracetam 150 mg/kg + Piracetam 300 mg/kg

* *

-50% -47.4%

* *

* -52.7%

3

*

0

Fig. 2 The effect of treatment with vinpocetine (1.5, 3 or 6 mg/kg), piracetam (150 or 300 mg/kg) on the brain tissue (cortex, hippocampus and striatum) levels of total antioxidant capacity (TAC: lmol/g issue) in rats given an intracerebral injection of the demyelinating agent ethidium bromide (10 ll of 0.1%). TAC is decreased by ethidium bromide in cortex, hippocampus and striatum. TAC is decreased in the hippocampus and striatum by the highest dose of vinpocetine and decreased in all brain areas by either dose of

piracetam. Data are expressed as the mean ± SE. n = 5. The percent change from ethidium bromide control group is shown on top of bars. Data were analyzed by one-way analysis of variance, followed by a Tukey’s multiple range test for post hoc comparison of group means. Effects with a probability of P \ 0.05 were considered to be significant. Asterisks indicate significant change from ethidium bromide control group

decrease in GSH by 12.7% was registered in the striatum (7.82 ± 0.54 vs. 8.96 ± 0.62 lg/g, P [ 0.05). The administration of vinpocetine at 1.5, 3 or 6 mg/kg increased GSH level over the ethidium bromide control values in all brain areas examined. GSH increased in the cortex by 24.3, 28.8 and 29.1% by 1.5, 3 and 6 mg/kg vinpocetine, respectively. It increased in the hippocampus by 54.8, 39.1 and 20.2% by 1.5, 3 and 6 mg/kg vinpocetine, respectively. GSH increased in the striatum by 23, 23.8 and 19.7% by 1.5, 3 and 6 mg/kg vinpocetine, respectively. All the increments in GSH by vinpocetine were statistically significant when compared with the ethidium bromide control group (P \ 0.05). In the hippocampus, the increment in GSH was less marked as the dose of the drug increased i.e., the low dose was associated with the most marked increase in GSH. Reduced glutathione increased in hippocampus and striatum by the highest dose of piracetam (by 20.8 and 16.9%, respectively vs. ethidium bromide control value (8.12 ± 0.58 vs. 6.72 ± 0.41 lg/g, P \ 0.05 and 9.14 ± 0.56 vs. 7.82 ± 0.53 lg/g, P \ 0.05, respectively). Thus ethidium bromide decreased GSH in both the cortex and hippocampus. GSH is increased by vinpocetine in all brain areas and increased in the hippocampus and striatum by the highest dose of piracetam (Fig. 3).

(38.9 ± 2.4 vs. 29.5 ± 1.9 lmol/l, P \ 0.05). In ethidium bromide treated rats, serum nitric oxide decreased after treatment with 1.5, 3 or 6 mg/kg vinpocetine by 36.8% (24.6 ± 1.8 lmol/l, P \ 0.05), 35% (25.1 ± 2.0 lmol/l, P \ 0.05) and 34.7% (25.4 ± 1.8 lmol/l, P \ 0.05) compared with the ethidium bromide control group (38.9 ± 2.4 lmol/l). The inhibition of nitric oxide by vinpocetine was not dose-dependant. The administration of piracetam at 150 or 300 mg/kg had no significant effect on serum nitric oxide level. These results suggest that nitric oxide in serum is increased by ethidium bromide but decreased after vinpocetine treatment (Fig. 4).

Serum Nitric Oxide Ethidium bromide resulted in an increased serum nitric oxide by 31.9% compared with the saline-treated group

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Brain Glucose Brain tissue glucose was unchanged by ethidium bromide treatment. Glucose is increased in the cortex and hippocampus of ethidium bromide treated rats, following 3 or 6 mg/kg vinpocetine. It increased by 31.5–23.8% after 3 mg/kg vinpocetine (167 ± 5.2 vs. 127 ± 3.8 lg/g, P \ 0.05 in cortex and 246.7 ± 7.1 lg/g vs. 195.3 ± 5.8 lg/g, P \ 0.05 in hippocampus). It increased by 23.2–22% after 6 mg/kg vinpocetine (156.5 ± 4.9 vs. 127 ± 3.8 lg/g, P \ 0.05 in cortex and 238.3 ± 6.2 lg/g vs. 195.3 ± 5.8 lg/g, P \ 0.05 in hippocampus). Glucose decreased in all brain areas after piracetam administration. Glucose in brain decreased by 25.6, 37 and 38.5% in the cortex, hippocampus and striatum, respectively by 150 mg/kg piracetam (94.3 ± 2.6 vs. 127 ± 3.8 lg/g,

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Reduced glutathione (µg/g tissue)

12 11

Cortex

10

28.8% 24.3% 29.1% * * *

9

Striatum

54.8% * 39.1% *

20.2% 20.8% * *

23.8% 23% 19.7% 16.9% * * * *

8 7

Saline Ethidium control + Vinpocetine 1.5 mg/kg + Vinpocetine 3 mg/kg + Vinpocetine 6 mg/kg + Piracetam 150 mg/kg + Piracetam 300 mg/kg

6 5 4 3 2 1 0

Fig. 3 The effect of treatment with vinpocetine (1.5, 3 or 6 mg/kg), piracetam (150 or 300 mg/kg) on the brain tissue (cortex, hippocampus and striatum) concentrations of reduced glutathione (GSH: lg/g tissue) in rats given an intracerebral injection of the demyelinating agent ethidium bromide (10 ll of 0.1%). GSH is decreased by ethidium bromide in cortex and hippocampus. It increased by vinpocetine in all brain areas and increased in the hippocampus and

striatum by the highest dose of piracetam. Data are expressed as the mean ± SE. n = 5. The percent change from ethidium bromide control group is shown on top of bars. Data were analyzed by oneway analysis of variance, followed by a Tukey’s multiple range test for post hoc comparison of group means. Effects with a probability of P \ 0.05 were considered to be significant. Asterisks indicate significant change from ethidium bromide

45 Saline

Nitric oxide (µmole/ L)

40

Ethidium control

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35 30

-36.8%

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+ Vinpocetine 1.5 mg/kg

-35.5%

-34.7%

+ Vinpocetine 3 mg/kg

*

*

+ Vinpocetine 6 mg/kg

25 20

+ Piracetam 150 mg/kg + Piracetam 300 mg/kg

15 10 5 0

Fig. 4 The effect of treatment with vinpocetine (1.5, 3 or 6 mg/kg), piracetam (150 or 300 mg/kg) on serum nitric oxide level (lmol/l) in rats given an intracerebral injection of the demyelinating agent ethidium bromide (10 ll of 0.1%). Nitric oxide in serum is increased by ethidium bromide but decreased after vinpocetine treatment. Data are expressed as the mean ± SE. n = 5. The percent change from

ethidium bromide control group is shown on top of bars. Data were analyzed by one-way analysis of variance, followed by a Tukey’s multiple range test for post hoc comparison of group means. Effects with a probability of P \ 0.05 were considered to be significant. Asterisks indicate significant change from ethidium bromide control group

P \ 0.05 in cortex, 123 ± 2.8 vs. 195.3 ± 5.8 lg/g, P \ 0.05 in hippocampus and 186 ± 2.9 vs. 302.5 ± 5.3 lg/g, P \ 0.05 in striatum). It decreased by 26.4, 38.2 and 39.6% in the cortex, hippocampus and striatum, respectively by 300 mg/kg piracetam (93.5 ± 2.2 vs. 127 ± 3.8 lg/g, P \ 0.05 in cortex, 120.6 ± 2.4 vs. 195.3 ± 5.8 lg/g, P \ 0.05 in hippocampus and 182.5 ± 2.6 vs. 302.5 ± 5.3 lg/g, P \ 0.05 in striatum). The above findings indicate that brain tissue glucose was unchanged by ethidium bromide treatment. Glucose is increased in the cortex and hippocampus by 3 or 6 mg/kg vinpocetine but decreased in all brain areas after piracetam administration (Fig. 5).

Acetylcholinesterase Activity AChE activity was unchanged in the cortex (4.46 ± 0.42 vs. 4.32 ± 0.66 lmol SH/g/min) or hippocampus (3.28 ± 0.32 vs. 3.07 ± 0.84 lmol SH/g/min) but was markedly increased by 84.9% in striatum (12.46 ± 0.54 vs. 6.74 ± 0.62 lmol SH/g/min, P \ 0.05) after ethidium bromide injection. AChE activity increased in the cortex after vinpocetine 1.5, 3 and 6 mg/kg by 37.7, 43.5 and 68.4% compared with the ethidium bromide control group (6.14 ± 0.54, 6.4 ± 0.58 and 7.51 ± 0.66 vs. 4.46 ± 0.42 lmol SH/g/min, P \ 0.05) and by compared with the saline-treated

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Neurochem Res (2011) 36:1062–1072 Striatum 350

Hippocampus

300

Glucose (µg/ g tissue)

22%

*

250

Cortex 200

* 150

-35.8%

*

23.2%

*

-37% -38.2% -25.6% -26.4%

100

-39.6%

26.3%

31.5%

*

Saline Ethidium control + Vinpocetine 1.5 mg/kg + Vinpocetine 3 mg/kg + Vinpocetine 6 mg/kg + Piracetam 150 mg/kg + Piracetam 300 mg/kg

*

* *

* *

50 0

Fig. 5 The effect of treatment with vinpocetine (1.5, 3 or 6 mg/kg), piracetam (150 or 300 mg/kg) on the brain tissue (cortex, hippocampus and striatum) concentrations of glucose (lg/g tissue) in rats given an intracerebral injection of the demyelinating agent ethidium bromide (10 ll of 0.1%). Brain tissue glucose was unchanged by ethidium bromide treatment. Glucose is increased in the cortex and hippocampus by 3 or 6 mg/kg vinpocetine. Glucose decreased in all

brain areas after piracetam administration. Data are expressed as the mean ± SE. n = 5. The percent change from ethidium bromide control group is shown on top of bars. Data were analyzed by oneway analysis of variance, followed by a Tukey’s multiple range test for post hoc comparison of group means. Effects with a probability of P \ 0.05 were considered to be significant. Asterisks indicate significant change from ethidium bromide control group

group). AChE increased in the cortex by 82.1% after piracetam at 300 mg/kg (8.12 ± 0.44 vs. 4.46 ± 0.42 lmol SH/g/min, P \ 0.05). It increased in the hippocampus after 1.5 or 3 mg/kg vinpocetine by 68.9 (P \ 0.05) and 16.8% (P \ 0.05) and increased by 33.5% (P \ 0.05) after piracetam at 150 mg/kg compared with the ethidium bromide control group. In the striatum, AChE activity showed further increase by 1.5 or 3 mg/kg vinpocetine by 45.2 and 18.9% (18.09 ± 0.66 and 14.81 ± 0.64 vs. 12.46 ± 0.54 lmol SH/g/min, P \ 0.05) but decreased after the highest dose of the drug by 28.4% (P \ 0.05) compared with the ethidium bromide control group. It increased by 49.6% (P \ 0.05) after 150 mg/kg piracetam, but decreased by 37.4% (P \ 0.05) by 300 mg/kg piracetam compared with the ethidium bromide control group (Fig. 6). Values of AChE activity were: 18.64 ± 0.58 and 7.80 ± 0.56 for 150 and 300 mg/kg of piracetam, respectively versus ethidium bromide (positive control) value of 12.46 ± 0.46 lmol SH/g/min. In summary, AChE activity increased in cortex, hippocampus and striatum after 1.5 or 3 mg/kg vinpocetine, The highest dose (6 mg/kg) increased AChE in the cortex but decreased it in striatum. It increased in the hippocampus and striatum after piracetam at 150 mg/kg. The highest dose of piracetam increased AChE in the cortex but decreased it in striatum.

rat brain is associated with increased oxidative burden. The oxidative stress induced by ethidium bromide is reflected in increased production of thiobarbituric acid (TBA)-MDA adducts, decreased total antioxidant capacity, decreased reduced glutathione in brain and increased serum nitric oxide levels. AChE activity increased in striatum as well. Increased oxidative stress can lead to deleterious effects on neuronal function and has been linked to neurodegenerative diseases as well as to age-related cognitive deficits [36, 37]. It was therefore, thought pertinent to examine the possible modulatory effect of two commonly used memory enhancing and neuroprotective drugs namely, vinpocetine and piracetam in this model of oxidative brain injury. The level of tissue MDA adducts was measured as a marker of free radical-induced macromolecular damage. Ethidium bromide resulted in significant elevation of MDA in cortex and striatum. The increase in MDA in the striatum and in cortex was decreased by the lower dose of either vinpocetine or piracetam, respectively, suggesting an antioxidant effect. In contrast, MDA increased in the cortex and hippocampus and in cortex, hypothalamus and striatum by the higher dose of vinpocetine or piracetam, respectively, suggesting an increased free radical production with the higher dose of either nootropic. Glutathione (GSH) is an intracellular tripeptide (glycyl– glutamic acid–cysteine) common in all tissues, which provides the major antioxidant defense mechanism [38]. Glutathione is the most abundant sulfhydryl-containing compound in the cells and the brain’s most important cellular free radical scavenger [39]. Free glutathione is present mainly in its reduced form (GSH) which is

Discussion The present study provided the evidence that demyelination due to the local injection of ethidium bromide into the

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1069 Striatum 45.2%

* Acetylcholinesterase (µmol SH/g/min)

18

Saline Ethidium control + Vinpocetine 1.5 mg/kg + Vinpocetine 3 mg/kg + Vinpocetine 6 mg/kg + Piracetam 150 mg/kg + Piracetam 300 mg/kg

18.9%

16

49.6%

*

*

14 12

Cortex -28.4%

10 8 6 4

68.4% 43.5% 37.7%

*

*

*

82.1%

Hippocampus

*

*

-37.4%

* 68.9%

*

33.5% 16.8%

*

* +

2 0

Fig. 6 The effect of treatment with vinpocetine (1.5, 3 or 6 mg/kg), piracetam (150 or 300 mg/kg) on the brain tissue (cortex, hippocampus and striatum) acetylcholinesterase activity (AChE: lmol SH/g/ min) in rats given an intracerebral injection of the demyelinating agent ethidium bromide (10 ll of 0.1%). Vinpocetine and piracetam displayed variable effects on regional AChE activity. AChE activity increased in cortex, hippocampus and striatum after 1.5 or 3 mg/kg vinpocetine, The highest dose increased AChE in the cortex and decreased it in striatum. It increased in the hippocampus and striatum

after piracetam at 150 mg/kg. The highest dose increased AChE in the cortex and decreased it in striatum. Data are expressed as the mean ± SE. n = 5. The percent change from ethidium bromide control group is shown on top of bars. Data were analyzed by oneway analysis of variance, followed by a Tukey’s multiple range test for post hoc comparison of group means. Effects with a probability of P \ 0.05 were considered to be significant. Asterisks indicate significant change from ethidium bromide control group

converted into oxidized glutathione (GSSG) during oxidative conditions. Reduced levels of GSH have been associated with a number of neurological diseases, suggesting an increase of oxidative stress in these conditions [40]. In the present work, ethidium bromide resulted in a significant decrease of reduced glutathione in both the cortex and hippocampus. Both vinpocetine and piracetam offered significant protection against GSH depletion induced by ethidium bromide. Significant increases in GSH were observed after vinpocetine in all brain areas and in hippocampus and striatum after the highest dose of piracetam. It is to be noted, however, that in case of vinpocetine, with the higher dose of the drug, the increment in GSH was less apparent in the hippocampus and striatum. This occurred along with decreasing total antioxidant capacity as well as an increased malondialdehyde in the cortex and hippocampus. These findings are likely to suggest a pro-oxidant effect as well for vinpocetine at 6 mg/kg and explaining the less marked increase in GSH compared with the lower dose of the drug. In case of piracetam, the highest dose of the drug increased lipid peroxidation in brain, whilst eliciting increased brain GSH. An intriguing explanation for these observations is that at their high concentration, these drugs exhibit pro-oxidant properties and increase free radical production or act as a free radical and in this latter case, it is possible that either piratcetam or vinpocetine react with other free radicals or antioxidant

systems other than glutathione, which is spared in this condition. Nitric oxide production can be estimated from determining the concentrations of nitrite and nitrate end products [35]. Within the central nervous system, nitric oxide is an important physiological signaling molecule, but when generated in excess, neurotoxicity might ensue [41]. Toxic demyelination was greatly prevented in mice lacking neuronal NOS (but not eNOS_/_mice) with a dramatic increase in mature oligodendrocyte survival and a decrease in apoptosis [42]. Multiple sclerosis patients in stable and acute phase like subjects with other neurological diseases showed high levels of nitric oxide in cerebrospinal fluid compared to the control group [43]. In addition nitric oxide production by peripheral blood leukocytes was higher in multiple sclerosis compared to healthy controls, independent of the disease course [44]. Nitric oxide is generated by inflammatory cytokines due to the action of inducible nitric oxide (iNOS) [45]. In the present study, nitric oxide production was markedly raised in ethidium bromide-treated rats, as evidenced by an increased nitric oxide metabolites (nitrite/nitrate concentration) in serum, thereby, suggesting increased iNOS expression. Treatment with vinpocetine significantly attenuated the ethidium bromide-induced increase in serum nitric oxide level, though not in a dosedependent manner, possibly due to a decrease in the inflammatory response caused by the toxic agent. Nitric

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oxide levels were unaltered by piracetam treatment. In dogs subjected to hemorrhagic shock, piracetam enhanced recovery in brain damage without altering nitric oxide levels [46]. In the present study, total antioxidant capacity was decreased by ethidium bromide in the cortex, hippocampus and striatum, thereby, suggesting consumption of antioxidant mechanisms by reactive oxygen species. TAC showed further reduction in the hippocampus and striatum by the highest dose of vinpocetine and by either dose of piracetam, thereby suggesting a pro-oxidant effect for these agents. In the case of piracetam, this reduction was more marked with the lower dose of the drug. The concept of a single test that might reflect total antioxidant capacity has been recently introduced as an indication of oxidative status in body fluids or tissues [47]. A decrease in plasma total antioxidant capacity was observed in patients with multiple sclerosis [37]. Low total antioxidant capacity could be indicative of oxidative stress or increased susceptibility to oxidative damage. It does not, however, appear to reflect the sum of activities of different antioxidant defense mechanisms e.g., antioxidant enzymes and glutathione [48]. It has also been suggested that information on antioxidant capacity by itself is not sufficient to make inferences about oxidative stress and that a marker of antioxidant capacity should always be associated with at least a marker of oxidative damage when the aim is to make inferences about oxidative stress [49]. The mechanism by which the high doses of piracetam or vinpocetine increase MDA and decrease TAC in brain homogenates is not clear. Piracetam facilitates learning and retrieval of information and protect the brain from physical and chemical noxious agents [50]. The mode of action of piracetam is, however, not obvious and such mechanisms as increased membrane fluidity, improved erythrocyte deformability, normalization of hyperactive platelet aggregation, mitochondrial membrane stabilization and protection has been suggested to account for its effect on memory [51–53]. In spinal cord ischemia–reperfusion injury in the rabbit, piracetam has been reported to suppress malondialdehyde, increase glutathione peroxidase activity and decrease xanthine oxidase level [25], thereby suggesting an antioxidant effect for the drug. For vinpocetine, increased cerebral blood flow and the consequent increase in the regional cerebral glucose uptake has been postulated to account for the beneficial effect of the agent on memory function in the aged individuals [18]. Vinpocetine (100 lM) inhibited the ascorbate/Fe2? stimulated consumption of oxygen and thiobarbituric acid reactive substances accumulation in rat brain synaptosomes, in a concentration-dependent manner [21]. Vinpocetine at concentration of 40 lM blocked the inhibition of the mitochondrial respiratory chain complexes II–III and

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IV and completely abolished the depletion of pyruvate levels induced by toxic concentrations of Abeta peptides [26]. In cell cultures, 1 mM piracetam or 0.1 lM vinpocetine, protected astrocytes during hypoxia. The higher concentration of vinpocetine (10 lM) was, however, detrimental in hypoxic conditions [54]. Vinpocetine increases DOPAC by impairing the vesicular storage of dopamine [55]. Piracetam has been reported to increase dopamine in cortics and striatium [56–58] which might increase free radical production. Dopamine is subject to auto-oxidation to semiquinone and quinones generating free radicals such as superoxide anion, peroxynitrite, hydroxyl radical and nitrate radical. Semiquinones and quinines themselves can form conjugates with thiol compounds such as GSH and L-cysteine, and generate other radicals that are also toxic [59]. It is therefore possible that at high doses, both vinpocetine and piracetam might increase free radical production through their effect on dopamine level in brain. The study also demonstrated an increased glucose concentration in both the cortex and striatum by the vinpocetine at 3 and 6 mg/kg, in line with other studies that had shown increased brain glucose availability by the agent [18]. Being a potent vasodilator at the cerebral vascular bed, the drug increases cerebral blood flow that increases the regional cerebral glucose uptake and, to a certain extent, glucose metabolism in the so-called peri-stroke region as well as in the relatively intact brain tissue [18]. Studies indicated that vinpocetine, administered intravenously in humans, readily passes the blood–brain barrier, showing high uptake in the brain structures corresponding to those in which vinpocetine has been shown to induce elevated metabolism and blood flow [60]. In contrast to vinpocetine, results of the present study showed decreased glucose concentration in all brain areas by piracetam administration. Other studies suggested that the drug increases local cerebral glucose utilization [61]. The regional depressions in glucose metabolism observed following scopolamine treatment in the rat hippocampus were completely reversed by piracetam (100 mg/kg) [62]. In Alzheimer’s disease (though not in multi-infarct dementia/unclassified dementia), i.v. administration of piracetam, for 2 weeks, significantly improved regional glucose metabolism in most cortical areas [63]. Acetylcholinesterase is important in cellular stress responses [64, 65]. AChE activity in the neocortex and hippocampus, but not cerebellum, of animals exposed to a single stress session increased by two to threefold within 50 min after stress and cortical activity remained significantly higher than that in control mice for over 80 h [64]. Transcription and release of AChE by reactive astroglia is increased in response to oxidative stress. These results indicated that, while basal AChE release is constitutive and nonspecific, induced AChE release is part of a generalized

Neurochem Res (2011) 36:1062–1072

cellular response to oxidative stress specifically regulated by Ca?? influx through L-VGCC [66]. In the present study, AChE activity was markedly increased in striatum 6 days after ethidium bromide injection. Other researchers reported inhibition of AChE activity in cortex, striatum, hippocampus and cerebellum 15–30 days after injection of ethidium bromide [28]. In the current study, AChE activity showed further increase by 1.5–3 mg/kg vinpocetine in all brain areas. The higher dose of 6 mg/kg increased AChE activity in cortex, but decreased it in striatum. AChE activity increased by 150 mg/kg piracetam in hippocampus and striatum. The higher dose of the drug increased AChE activity in cortex, but decreased it in striatum; findings which appear to correlate with the increase in MDA and decrease in TAC by either drug. The significance of these observations is yet to be determined, for the striatum is an area vital for control of voluntary motor activity, where parkinsonian symptoms has been theorized to be a result of an imbalance between cholinergic and dopaminergic neuronal activity and where anticholinergic medications have a role in control of Parkinson’s tremor [67]. In summary, findings in the present study indicates that demyelination due to the local injection of ethidium bromide into the rat brain is associated with increased oxidative stress. The study suggests an antioxidant effect for the nootropic drugs vinpocetine and piracetam at low doses. In contrast, the higher doses were associated with an increase in oxidative stress, though both drugs offered significant protection against GSH depletion induced by ethidium bromide. The clinical significance of these findings needs to be determined.

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