Protoplasma

, Volume 250, Issue 5, pp 1067–1078 | Cite as

Attenuation of oxidative damage-associated cognitive decline by Withania somnifera in rat model of streptozotocin-induced cognitive impairment

  • Md. Ejaz Ahmed
  • Hayate Javed
  • Mohd. Moshahid Khan
  • Kumar Vaibhav
  • Ajmal Ahmad
  • Andleeb Khan
  • Rizwana Tabassum
  • Farah Islam
  • Mohammed M. Safhi
  • Fakhrul Islam
Original Article

Abstract

Oxidative stress is a critical contributing factor to age-related neurodegenerative disorders. Therefore, the inhibition of oxidative damage, responsible for chronic detrimental neurodegeneration, is an important strategy for neuroprotective therapy. Withania somnifera (WS) extract has been reported to have potent antioxidant and free radical quenching properties in various disease conditions. The present study evaluated the hypothesis that WS extract would reduce oxidative stress-associated neurodegeneration after intracerebroventricular injection of streptozotocin (ICV-STZ) in rats. To test this hypothesis, male Wistar rats were pretreated with WS extract at doses of 100, 200, and 300 mg/kg body weight once daily for 3 weeks. On day 22nd, the rats were infused bilaterally with ICV-STZ injection (3 mg/kg body weight) in normal saline while sham group received only saline. Two weeks after the lesioning, STZ-infused rats showed cognitive impairment in the Morris water maze test. The rats were sacrificed after 3 weeks of the lesioning for the estimation of the contents of lipid peroxidation, reduced glutathione, and activities of glutathione reductase, glutathione peroxidase, and catalase. Pretreatment with WS extract attenuated behavioral, biochemical, and histological alterations significantly in dose-dependent manner in the hippocampus and cerebral cortex of ICV-STZ-infused rats. These results suggest that WS affords a beneficial effect on cognitive deficit by ameliorating oxidative damage induced by streptozotocin in a model of cognitive impairment.

Keywords

Antioxidant Cognitive impairment Oxidative stress Streptozotocin Withania somnifera 

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by deterioration of memory and cognitive function. It affects millions of people and has become a major medical and social burden for developing countries. AD is linked with progressive and irreversible loss of neurons, mainly in the cortex and hippocampus. The neuropathological hallmarks of AD include amyloid beta plaques and neurofibrillary tangles composed of the hyperphosphorylated tau protein (Tundo et al. 2012; Thompson and Vinters 2012).

Oxidative stress, an imbalance between prooxidant and antioxidant level, is known to contribute the pathogenesis of neurodegenerative disorders including AD (Butterfield 2004; Zhu et al. 2005). This imbalance may originate from an overproduction of free radicals or from a reduction in antioxidant defenses due to the high consumption of oxygen and polyunsaturated fatty acids (Halliwell and Gutteridge 1985; Halliwell 2001; Khan et al. 2009). Oxidative damage to lipid and protein (protein carbonyl formation) can lead to structural and functional disruption of the cell membrane, inactivation of enzymes and, finally, cell death. Thus, it can be speculated that supplemental antioxidant treatment may boost the system to stay normal against the oxidative stress.

Intracerebroventricular (ICV) injection of streptozotocin (STZ) in rats impairs brain glucose, energy metabolism, cholinergic transmission, and increases generation of free radicals, leading to cognitive impairment (Hoyer and Lannert 1999; Ishrat et al. 2009; Javed et al. 2011). Streptozotocin when administered ICV damages the septohippocampal system (Prickaerts et al. 1999) whereby memory impairment in rats could occur due to direct damage to the system. This is supported by reduced choline acetyltransferase (ChAT) activity in the hippocampus, (Blokland and Jolles 1994). Experimental intracerebroventricular administration of streptozotocin in rats has been shown to produce biochemical alterations similar to those observed in sporadic AD and therefore considered to be a valid experimental model. Since oxidative damage is concerned in the etiology of neurological complications including AD, treatment with antioxidants has been used as a therapeutic approach in neurodegenerative disease.

Withania somnifera (WS) (commonly known as ashwagandha) is used in many indigenous system of medicine, mainly Ayurveda in India (Ahmad et al. 2005; Sehgal et al. 2012). The known biological active chemical constituents of WS are Withaferin A and saponins (Mishra et al. 2000). WS has been reported to be a potent enhancer of cellular antioxidants and possesses a significant free radical scavenging activity in various disease models (Davis and Kuttan 2001; Bhattacharya et al. 2001). Withaferin-A, is the principal withanolide in Indian WS and highly oxygenated steroidal lactone, related to Solanaceae species. Its pharmacological properties includes anti-inflammatory, antitumor, and antioxidant (Sharada et al. 1996; Bhattacharya et al. 1997; Konar et al. 2011; Sehgal et al. 2012). Therefore, the present study was executed to test the antioxidant potential of characterized WS to ameliorate the cognitive impairment in the ICV-STZ model of rats.

Materials and methods

Quality control of Withania alcoholic extract

The authenticity and quality of W. somnifera extract was checked by fingerprinting and quantification of Withaferin A in the sample using the CAMAG HPTLC system.

A general alkaloid extraction procedure was followed for the extraction of Withaferin A from a sample. Four hundred milligrams of extract was taken into a 50-mL round bottom flask containing 40 mL of aqueous acidic solution (10 % HCl) and refluxed for 1 h. It was filtered and residue washed with fresh solvent. The combined washing and filtrate was cooled and basified by adding dilute ammonia solution to make the pH up to 10. This mixture was then extracted with chloroform using a separating funnel; the chloroform layer was separated and the process was repeated thrice for complete extraction. The combined chloroform extract was evaporated to dryness on water bath and reconstituted in 2 mL of ethanol.

The samples were spotted in the form of bands of a width of 5 mm with a Camag microliter syringe on precoated silica gel aluminium plate 60 F-254 (5 × 10 cm with 0.2 mm thickness, E. Merck, Germany) using a Camag Linomat V (Switzerland) sample applicator. A constant application rate of 120 nl/s was employed, and the space between two bands was 15 mm. The slit dimension was kept at 5 × 0.20 mm, and a 20-mm/s scanning speed was employed. The mobile phase consisted of toluene/ethyl acetate/formic acid (5:5:1). Linear ascending development was carried out in twin trough glass chambers saturated with the mobile phase. The optimized chamber saturation time for the mobile phase was 20 min at room temperature. The length of the chromatogram run was 80 mm. Subsequent to the development, TLC plates were dried in a current of air with the help of an air-dryer. Densitometric scanning was performed on a Camag TLC scanner IV in the absorbance mode at 214 nm. The source of radiation utilized was from a deuterium and tungsten lamp.

Animals and treatments

Male Wistar rats (1 year old) weighing 400 ± 20 g were obtained from the Central Animal House of Jamia Hamdard (Hamdard University), New Delhi, India. Rats were housed in groups of four animals per cage and had free access to food and water ad libitum. They were kept in the Central Animal House at an ambient temperature of 25 ± 2 °C and a relative humidity of 45–55 % with 12-h light/dark cycles. The food was withdrawn 12 h before the surgery. Experiments were conducted in accordance with the Animal Ethics Committee of Jamia Hamdard, approved by the Government of India.

Experiments were carried out to evaluate the pretreatment effect of 100, 200, and 300 mg/kg body weight of WS extract for 3 weeks (orally) to the STZ infusion on the content of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), and for the activities of antioxidant enzymes. The rats were randomly divided into eight groups of ten animals each. The first group served as sham (S) and vehicle (orally) was given; group 2 was the vehicle-treated ICV–STZ-infused group (L); group 3 was the L group pretreated with 100 mg/kg WS; group 4 was the L group pretreated with 200 mg/kg of WS (WS200 + L), and group 5 was the L group pretreated with 300 mg/kg of WS (WS300 + L). Group 6 was sham-operated and WS 100 mg/kg pretreated (WS100 + S); group 7 was WS 200 mg/kg pretreated (WS200 + S), and group 8 was WS 300 mg/kg pretreated (WS300 + S).

Intracerebroventricular injection of streptozotocin

The animals were anesthetized with chloral hydrates (400 mg/kg) intraperitoneally (i.p.) and placed on a stereotaxic frame (dual manipulator model 51600, Stoelting Co., IL, USA). The skin overlying the skull was incised to expose it, and the coordinates for the lateral ventricle (Paxinos and Watson 1986) were measured accurately as arterio-posterior −0.8 mm, lateral 1.5 mm, and dorso-ventral −4.0 mm relative to the bregma and ventral from the dura with the tooth bar set at 0 mm. A burr hole was made in the skull by an automatic microdrilling machine attached on a stereotaxic apparatus. Through the hole, a 28-gauge Hamilton® syringe of 10 μL attached to a micro-injector unit and piston of the syringe was lowered manually into each lateral ventricle. The lesion groups received a bilateral ICV injection of STZ (3 mg/kg body weight in saline, 5 μL/injection) (Ishrat et al. 2006; Javed et al. 2011). The sham groups underwent the same surgical procedures and the same volume of saline was injected instead of STZ. After surgery, the rats were housed individually and had access to food and water ad libitum.

Behavioral testing

The behavioral tests were started 2 weeks after ICV-STZ infusion. The experiment was performed between 9.00 a.m. to 4.00 p.m. at standard laboratory conditions. Behavioral tests were performed and analyzed by a research blinded to the experimental conditions.

The spatial learning and memory of animals were tested in a Morris water maze (Morris 1984). It consisted of a circular water tank (132-cm diameter and 60-cm height) that was filled 30 cm with water (25 ± 2 °C). A non-toxic white paint was used to render the water opaque. The pool was divided virtually into four equal quadrants, labeled north–south–east–west. An escape platform (10 cm in diameter) was hidden 2 cm below the surface of water on a fixed location in one of the four quadrants of the pool. The platform remained in the same quadrant throughout the experiment. Before the training started, the rats were allowed to swim freely into the pool for 60 s without a platform. They were given four trials (once from each starting position) per session for 5 days, each trial having a ceiling time of 60 s and a trial interval of approximately 30 s. After climbing on to the platform, the animal remained there for 30 s before the commencement of the next trial. If the rats failed to reach the escape platform within the maximum allowed time of 60 s, it was gently placed on the platform and allowed to remain there for the same interval of time. An overhead video camera was connected to a video monitor and a computer software was used to track the animal’s path and to calculate the escape latency and travelled distance (path length).

Preparation of samples for biochemical analysis

After 3 weeks of ICV-STZ infusion, the animals were sacrificed and their brains were taken out quickly on ice to dissect the hippocampus and cerebral cortex. The dissected brain parts were homogenized at 4 °C in 10 mM phosphate buffer (PB, pH 7.4) having 10 μL/mL protease inhibitor (5 mM leupeptin, 1.5 mM aprotinin, 2 mM phenylmethylsulfonyl fluoride, 3 mM peptastatin A, 10 mM EDTA, 0.1 mM EGTA, 1 mM benzamidine, and 0.04 % butylated hydroxytoluene). The homogenate was centrifuged at 800 g for 5 min at 4 °C to separate the nuclear debris. The supernatant 1 (S1) was used for the estimation of lipid peroxidation and acetylcholinesterase (AChE) activity. The remaining S1 was further centrifuged at 10,500 × g for 30 min at 4 °C to get the post-mitochondrial supernatant (PMS) which was used for estimation of reduced GSH and antioxidant enzymes. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Assay of TBARS content a marker of lipid peroxidation

TBARS content was estimated by the method of Utley et al. (1967) as modified by Khan et al. (2009). S1 (0.1 mL) was taken into a 15 × 100-mm test tube and incubated at 37 °C in a metabolic shaker for 1 h. An equal volume of homogenate was pipetted into a centrifuge tube and placed at 0 °C and marked at 0-h incubation. After 1 h of incubation, 0.45 mL of 5 % (w/v) chilled TCA and 0.45 mL 0.67 % TBA were added and centrifuged at 4,000 × g for 10 min after thoroughly mixing. Thereafter, the supernatant was transferred to other test tubes and placed in a boiling water bath for 10 min. The absorbance of pink color developed was measured at 535 nm. The TBARS content was calculated by using a molar extinction coefficient of 1.56 × 105 M−1 cm−1 and expressed as nanomoles of TBARS formed/hour/milligram protein.

Assessment of reduced glutathione

GSH content was measured using the method described by Jollow et al. (1974) with a slight modification. PMS was mixed with 4.0 % sulfosalicylic acid (w/v) in 1:1 ratio (v/v). The samples were incubated at 4 °C for 1 h and centrifuged at 4,000 × g for 10 min at 4 °C. The assay mixture contained 0.1 mL of supernatant, 1.0 mM DTNB, and 0.1 M phosphate buffer pH 7.4 in a total volume of 3.0 mL. The yellow color developed was read immediately at 412 nm. The GSH content was calculated as micromoles GSH per milligram protein, using a molar extinction coefficient of 13.6 × 103 M−1 cm−1.

Assays of antioxidant enzymes

Glutathione peroxidase (GPx) activity was determined by the method of Mohandas et al. (1984). The reaction assay consisted of phosphate buffer (0.05 M, pH 7.0), EDTA (1 mM), sodium azide (1 mM), glutathione reductase (1 EU/mL), glutathione (1 mM), NADPH (0.2 mM), hydrogen peroxide (0.25 mM), and 0.1 mL of PMS in the final volume of 2 mL. The disappearance of NADPH at 340 nm was recorded at room temperature. The enzyme activity was calculated as nanomole NADPH oxidized per minute per milligram protein by using a molar extinction coefficient of 6.22 × 103 M−1 cm−1.

Glutathione reductase (GR) activity was measured by the method of Carlberg and Mannervik (1975) as modified by Mohandas et al. (1984). The reaction mixture consisted of phosphate buffer (0.1 M, pH 7.6), NADPH (0.1 mM), EDTA (0.5 mM), and oxidized glutathione (1 mM) and 0.05 mL of PMS in a total volume of 1 mL. The enzyme activity was quantified at room temperature by measuring the disappearance of NADPH at 340 nm and calculated as nanomole NADPH oxidized per minute per milligram protein using a molar extinction coefficient of 6.22 × 103 M−1 cm−1.

Catalase activity was assayed by the method of Claiborne (1985). Briefly, the assay mixture consisted of 0.05 M phosphate buffer (pH 7.0), 0.019 M H2O2, and 0.05 mL PMS in a total volume of 3.0 mL. The change in absorbance was recorded at 240 nm. Catalase activity was calculated in terms of nanomole H2O2 consumed per minute per milligram protein using a molar extinction coefficient of 43.6 × 103 M−1 cm−1.

Acetylcholinesterase activity

AChE activity was determined by the method of Ellman et al. (1961). Briefly 2.6 mL of PB (0.1 M, pH 8.0), 40 μL of 0.075 M acetylthiocholine iodide and 0.1 mL of buffered Ellman’s reagent (10 mM DTNB, 15 mM NaHCO3) were mixed and allowed to incubate for 5 min at room temperature. An enzyme sample (40 μL) was added, and optical density was measured at 412 nm within 5 min. AChE activity was expressed as nanomole thiocholine formed per minute per milligram protein using a molar extinction coefficient of 13.6 × 103 M−1 cm−1.

Caspase-3 activity

Caspase-3 activity was measured with a kit (CasPASE TM colorimetric assay kit supplied with Ac-DEVD-AFC substrate) according to the manufacturer's instructions. Caspase-3 activity was calculated as nanomole per hour per milligram protein.

Hematoxylin and eosin staining

The animals were anesthetized with chloral hydrate on the 22nd day of lesioning and were perfused transcardially through the ascending aorta with 100 mL of ice cold phosphate-buffered saline (PBS 0.1 M, pH 7.4) followed by 4 % paraformaldehyde in cold PBS (0.1 M, pH 7.4). The brains were removed quickly, postfixed in the paraformaldehyde solution for 48 h and embedded with wax. Coronal sections having hippocampus 5-μm thickness were dewaxed and stained with hematoxylin and eosin (H&E). The sections were cleared in xylene and mounted in DPX mounting medium. Images were acquired using light microscopy (BX51, Olympus, Tokyo, Japan).

Immunohistochemistry

Immunohistochemistry was performed to detect the expression of ChAT protein. Coronal sections (5-μm thick) at the level of the hippocampus were dewaxed and processed immunohistochemical staining. Sections were collected serially on slides and placed in 3 % H2O2 in methanol for 20 min at room temperature to eliminate the endogenous peroxidase activity. The slides were washed with PBS for several times and pre-incubated in 1 % bovine serum albumin for 45 min at room temperature; thereafter, the slides were incubated with primary antibody, rabbit anti-ChAT (dilution, 1:200) at 4 °C overnight. The slides were washed with PBS to remove the unbound antibodies, and sections were incubated with goat anti-rabbit IgG (Jackson Immuno Research USA, dilution, 1:500) for 1 h at room temperature and avidin–biotin complex (ABC kit from Vector Laboratories Ltd., UK). The slides were treated with 3,4-diaminobenzidine (Vector Laboratories Ltd., UK) and observed under a light microscope. The positively stained cells for ChAT in the hippocampus lesions were counted in five randomized areas per rat brain. The results were expressed as the number of stained cells per square millimeter.

Statistical analysis

Results are expressed as mean ± S.E.M. A statistical analysis of the data was done by applying the analysis of variance, followed by Tukey's test. A P value < 0.05 was considered statistically significant.

Results

Quantification of Withaferin A from WS extract

The standard chromatograph shows peaks of Withaferin A at Rf 0.37 ± 0.02 (Fig. 1a, b). The peak area thus obtained with different concentration of standard were treated with linear least square regression to get the regression equation \( y=554.503+14.202\times \left( {{r^2}=0.9964} \right) \), which was used for quantification of Withaferin A in sample (in duplicate). The average of duplicate samples were taken and reported as the concentration of Withaferin A. It is found that withania extract contains 0.0232 % (w/w) of Withaferin A in the sample.
Fig. 1

HPTLC chromatogram of sample at 214 nm showing presence of Withaferin A at Rf 0.37 (a). Developed TLC plate showed the presence of Withaferin A in sample S2 (b). The Withaferin A was quantified as 0.0232 % (w/w) in extract

Effect of WS on performance in Morris water maze task

A decreased latency was observed in all groups of animals to find the platform from the second to the fifth day of experiment. However, lesion (L) group animals presented a significantly (p < 0.01) higher latency to find the platform than sham (S) group animals. The pretreatment with WS in WS100 + L, WS200 + L, and WS300 + L has shown a significant (p < 0.01) and dose dependent improvement in latency as compared to the L group (Fig. 2a).
Fig. 2

Effect of Withania somnifera (WS) on escape latency and path length in the Morris water maze test. Average escape latency time from day 2 to 5 to find submerged platform was significantly (p < 0.01) prolonged in lesion (L) group animals when compared to sham (S) group animals (A). Average distance travelled from day 2 to 5 to find the submerged platform was significantly (p < 0.01) prolonged in L group animals when compared to the S group animals (B). Pretreatment with WS has protected the learning deficits significantly (p < 0.01) in WS100 + L, WS200 + L, WS300 + L group animals as compared to L group animals. Values are expressed as mean ± S.E.M. (n = 10)

Decreased path length was shown by all the groups of animals to find the platform from the second to the fifth day of experiment. However, L group animals took a significantly (p < 0.01) longer distance (path length) to find the platform than the sham group animal. Pretreatment with WS in WS100 + L, WS200 + L, and WS300 + L has shown a significant (p < 0.01) and dose dependent improvement in path length as compared to the L group (Fig. 2b).

Effect of WS on TBARS content in the hippocampus and cerebral cortex

The effect of WS on the TBARS content was measured to demonstrate the oxidative damage to the membrane in the hippocampus (Fig. 3a) and cerebral cortex (Fig. 3b). The content of TBARS was significantly elevated (p < 0.05) in the L group as compared to the S group, and its content was significantly and dose dependently protected by the pretreatment with WS (p < 0.05, L vs. WS100 + L; p < 0.01, L vs. WS200 + L and WS300 + L). No significant change was observed between the WS pretreated sham group and S group.
Fig. 3

Effect of WS pretreatment on TBARS content in the hippocampus (A) and in cerebral cortex (B) of ICV-STZ-infused rats. TBARS content was significantly increased in the hippocampus and cerebral cortex of L group animals as compared to the S group (*p < 0.05 L Vs. S group). WS pretreatment significantly decreased the content of TBARS in the hippocampus and cerebral cortex of WS100 + L, WS200 + L, and WS300 + L group animals as compared to the L group animals (#p < 0.05 L vs. WS100 + L; ##p < 0.01, L vs. WS200 + L and WS300 + L). Values are expressed as mean ± S.E.M. (n = 10)

Effect of WS on glutathione content in the hippocampus and cerebral cortex

The content of GSH was significantly (p < 0.05) decreased in the hippocampus (Fig. 4a) and cerebral cortex (Fig. 4b) of the L group as compared to the S group. The level of GSH was protected significantly (p < 0.05, L vs. WS100 + L; p < 0.01, L vs. WS200 + L; p < 0.001, L vs. WS300) and dose dependently by WS. No significant change was observed between the WS pretreated sham group and S group.
Fig. 4

Effect of WS pretreatment on the content of GSH in the hippocampus (A) and cerebral cortex (B) of ICV-STZ infused rats. GSH level was significantly decreased in L group as compared to the S group (*p < 0.05 L vs. S group). WS pretreatment significantly restored the level of GSH in the hippocampus and cerebral cortex in WS100 + L, WS200 + L, WS300 + L group animals as compared to the L group animals (#p < 0.05 L vs. WS100 + L; ##p < 0.01, L vs. WS200 + L; and ###p < 0.001, L vs. WS300 + L). Values are expressed as mean ± S.E.M. (n = 10)

Effect of WS on the activities of antioxidant enzymes in the hippocampus and cerebral cortex

The activities of antioxidant enzymes (GPx, GR, and catalase) were decreased significantly in the L group as compared to the S group animals, and pretreatment with WS has significantly protected the activities of these enzymes dose dependently in the hippocampus and cerebral cortex (Tables 1 and 2). No significant change was observed between the WS pretreated sham group and S group
Table 1

Protective effect of W. somnifera on the activities of antioxidant enzymes in the hippocampus of ICV-STZ rats

Parameters

GPx (nmol NADPH oxidized min−1 mg−1 protein)

GR (nmol NADPH oxidized min−1 mg−1 protein)

Catalase (nmol H2O2 consumed min−1 mg−1 protein

Sham (S)

338.92 ± 13.8

356.5 ± 13.85

52.6 ± 3.8

Lesion (L)

146.6 ± 21.48* (−56.74 %)

144.5 ± 6.8* (−59.46 %)

17.2 ± 0.8* (−67.3 %)

WS100 + L

195.26 ± 19.59** (33.58 %)

185.7 ± 4.8** (28.5 %)

22.7 ± 1.19** (31.97 %)

WS200 + L

214.24 ± 7.56** (46.1 %)

201.2 ± 3** (39.2 %)

25.6 ± 1.60*** (48.8 %)

WS300 + L

259.46 ± 9.09*** (77.30 %)

212.4 ± 8.4*** (46.98 %)

27.9 ± 1.16*** (62.2 %)

WS100 + S

341.2 ± 7.05 (−6.72 %)

338.268 ± 14.68 (5.11 %)

52.8 ± 3.72 (−0.38 %)

WS200 + S

349.78 ± 14.50 (−3.2 %)

340.7 ± 20.06 (4.43 %)

53.0 ± 2.72 (−0.76 %)

WS300 + S

356.5 ± 13.85 (−5.18)

341.52 ± 8.7 (4.20 %)

56.4 ± 2.70 (−7.22)

Administration of WS significantly attenuated the activities of GPx, GR, and catalase in the hippocampus in WS100 + L, WS200 + L, and WS300 + L group animals as compared to L group animals

*p < 0.05, L vs. S group; **p < 0.05; ***p < 0.01, WS100 + L, WS200 + L, WS300 + L vs. L group. Values are expressed as mean ± S.E.M. (n = 10)

Table 2

Protective effect of WS on the activities of antioxidant enzymes in the cerebral cortex of ICV-STZ rats

Parameters

GPx (nmol NADPH oxidized min−1 mg−1 protein)

GR (nmol NADPH oxidized min−1 mg−1 protein)

Catalase (nmol H2O2 consumed min−1 mg−1 protein

Sham (S)

340.34 ± 11.39

478.23 ± 28.5

38.25 ± 2.7

Lesion (L)

152.57 ± 5.0* (−55.17 %)

282.9 ± 16.2* (−40.8 %)

18.57 ± 2.5* (−51.45 %)

WS100 + L

198.33 ± 11.7** (29.99 %)

319.9 ± 13.2 (13.0 %)

23.98 ± 2.6** (29.13 %)

WS200 + L

216.24 ± 7.1** (41.73 %)

352.7 ± 12.1** (24.6 %)

26.288 ± 1.1** (41.51 %)

WS300 + L

231.13 ± 3.9*** (51.49 %)

388.05 ± 20.7** (37.16 %)

29.84 ± 2.1*** (60.06 %)

WS100 + S

340.98 ± 4.5 (−0.18 %)

479.67 ± 8.1 (−0.30 %)

39.62 ± 3.6 (−3.58)

WS200 + S

340.91 ± 4.08 (−0.16 %)

478.31 ± 49.93 (−0.016)

38.16 ± 1.7 (0.23 %)

WS300 + S

342.35 ± 5.07 (−0.59 %)

486.31 ± 52.0 (−1.6 %)

39.94 ± 4.6 (−4.41 %)

Administration of WS significantly attenuated the activities of GPx, GR, and catalase in cerebral cortex in WS100 + L, WS200 + L, and WS300 + L group animals as compared to L group animals. Values are expressed as Mean ± S.E.M (n = 10)

*p < 0.05, L vs. S group; **p < 0.05; ***p < 0.01, WS100 + L, WS200 + L, WS300 + L vs. L group

Effect of WS on acetylcholinesterase activity in the hippocampus

The activity of AChE was increased significantly in the L group as compared to the S group, and pretreatment with WS has significantly protected the activity of AChE enzyme dose dependently in the hippocampus (Fig. 5). No significant change was observed between the WS pretreated sham group and S group.
Fig. 5

Effect of WS pretreatment on the activity of AChE in the hippocampus of ICV-STZ-infused rats. AChE activity was significantly increased in L group as compared to S group (*p < 0.05 L vs. S group). WS pretreatment has restored its activity significantly in WS100 + L, WS200 + L, and WS300 + L group animals as compared to the L group animals (#p < 0.05 L vs. WS100 + L; ##p < 0.01 L vs. WS200 + L; and ###p < 0.001, L vs. WS300 + L). Values are expressed as mean ± S.E.M (n = 10)

Effect of WS on caspases-3 activity

A significantly increased caspases-3 activity was observed in the hippocampus and cerebral cortex in the L group animals as compared to the S group. WS supplementation has significantly (p < 0.05) decreased the caspases-3 activity in the WS300 + L group animals when compared with the L group (Fig. 6). However, no significant alteration was observed in the WS pretreated sham group as compared to the S group (data not shown).
Fig. 6

Effect of WS pretreatment on caspase-3 activity in the hippocampus and cerebral cortex. Activity of caspase-3 was significantly increased in the hippocampus and cerebral cortex in the L group rats as compared to the S group rats. WS pretreatment significantly decreased the caspase-3 activity in the WS300 + L group rats as compared to L group rats. Values are expressed as mean ± SEM (n = 6). *p < 0.01, S vs. L group; #p < 0.05, WS300 + L vs. L group

Hematoxylin and eosin staining

Normal neuronal morphology with distinct cytoplasm and prominent nucleoli were observed in the sham group (Fig. 7a) animals. In the lesion group animals, a photomicrograph shows vacuolation as the degenerative changes in the CA1 region of the hippocampus (white arrow); however, the green arrow shows the degenerative changes in the neurons in the hippocampus of the CA1 region. WS pretreatment ameliorated the hippocampal neuronal abnormalities in the WS300 + L group (Fig. 7c) animals as compared to L group animals (Fig. 7). No significant change was observed between the WS pretreated sham group and S group (data not shown).
Fig. 7

Representative photomicrograph showing H&E staining in the CA1 region of the hippocampus. Black arrows indicate the normal pyramidal neuron in S group (a) and white arrows indicate the degenerated pyramidal neuron in L group (b) while L group pretreated with WS extract (300 mg/kg) has shown normal pyramidal neuronal staining (c). ×40 magnification

Effect of WS on ChAT expression

Cholinergic deficiency is supported by the reduced expression of ChAT in the CA1 hippocampal region of the brain. A reduced expression of ChAT in the L group animals was observed as compared to the S group animals. Pretreatment with WS restores the ChAT expression in the W300 + L group animals (Fig. 8c) as compared to the L group animal (Fig. 8). No significant change was observed between the WS pretreated sham group and S group (Data not shown).
Fig. 8

Representative photomicrograph illustrating the expression of ChAT in the CA1 region of the hippocampus (a). Prominent ChAT expression was observed in the S group animals (a); however, the L group animals showed a lower expression of ChAT (b) compared to the S group. Considerably more ChAT expressions were found in the WS pretreated group animals (c) as compared to the L group animals (×40 magnification) (b). Quantification of ChAT positive neurons (*p < 0.01, S vs. L groups; #p < 0.05 L vs. WS300 + L groups)

Discussion

The present study examined the pretreatment effects of standardized W. somnifera (WS) on cognitive deficits, oxidative stress, and histopathological changes in intracerebroventricular injection of streptozotocin (ICV-STZ)-induced memory deficits in an experimental model of cognitive impairment. It is well documented that the ICV-STZ rodent model is an appropriate animal model used for the study of cognitive impairment (Hoyer et al. 1991; Lannert and Hoyer 1998; Agrawal et al. 2009; Ishrat et al. 2006; Javed et al. 2011). In our study, WS pretreatment significantly alleviated the cognitive deficits, biochemical, and histopathological alterations in ICV-STZ-infused rats. The neuroprotective potential of WS suggests that it is a powerful antioxidant, corroborating previous reports (Ahmad et al. 2005; Konar et al. 2011; Sehgal et al. 2012). Our result showed a moderate amount of Withaferin A (0.0232 % w/w) is found to be present in the extract. Withaferin A is reported to have a potential antioxidant property in different types of oxidative stress-associated disease (Grover et al. 2010).

The Morris water maze (Withaferin A) test was used to evaluate the spatial learning and memory deficit in rats. A decreased escape latency and path length in Withaferin A task in repeated trials demonstrated intact learning and memory function in the animals pretreated with WS. ICV-STZ-infused rats have shown a significantly elevated escape latency and path length as compared to the sham group. While a significantly decreased time and distance travelled to reach the hidden platform was observed dose dependently in WS pretreated group. The data have shown the conformity of memory impairment indicating the beneficial effect of WS in ameliorating the cognitive deficits induced by ICV-STZ. Our findings are in agreement with other findings where WS attenuated the behavioral deficits in the animal model of cognitive impairment (Naidu et al. 2006; Soman et al. 2012).

Oxidative stress refers to the cytological consequences of imbalance between the production of free radicals and the ability of the cells to defend against them. This imbalance results in a buildup of oxidatively modified molecules that can cause cellular dysfunction and neuronal death. Under normal conditions, an array of endogenous cellular defense system exists to counter balance the reactive oxygen species (Halliwell and Gutteridge 1985; Liu et al. 2003). The antioxidant system requires reduced GSH, a tripeptide and an essential antioxidant, which is responsible to buffer the free radicals in the brain tissue (Meister 1988). It eliminates H2O2 and organic peroxides by glutathione peroxidase (GPx) and catalase (Sun 1990). During free radical clearance, oxy radicals are reduced by GPx at the cost of GSH to form glutathione disulphide (GSSG). GSH is further produced by redox recycling, in which GSSG is reduced to GSH by glutathione reductase with an expenditure of one NADPH molecule. A reduced level of GSH impairs H2O2 clearance and endorses the formation of OH radical, the most toxic molecule to the brain, leading to a higher free radical level and more oxidative stress (Freeman and Crapo 1982; Meister 1988; Sun 1990). Lipid peroxidation (LPO) indicates neuronal membrane degeneration and reported early AD brain (Blokland and Jolles 1994). There are several reports about the modulatory effect of WS on LPO and antioxidant enzymes (Mishra et al. 2000; Ahmad et al. 2005; Konar et al. 2011). In agreement with these findings, we also found that WS significantly reduced the TBARS content and increased the activities of antioxidant enzymes along with the GSH level in the hippocampus and in the cerebral cortex following STZ infusion. The neuroprotective effect of WS could be attributed to its potential antioxidant effect suggesting that STZ-induced learning and memory impairment is associated with oxidative stress.

The histological examination provides for evaluating neural damage and the treatment effect. In the present study, we observed marked morphological changes in the hippocampus of ICV-STZ-induced rats. It is interesting that the degree of suppression of histopathologic lesions in the brain by WS roughly parallels the degree of suppression of behavioral and biochemical parameters by WS, and this finding corroborates the efficacy of WS in this model. Moreover, the effects of WS on ICV-STZ-induced rats are almost similar to those of curcumin, naringenin, and s-allyl cystein (Ishrat et al. 2009; Javed et al. 2011; Khan et al. 2012). Thus, these results indicate that WS could be an effective approach for attenuating the STZ-induced cognitive deficits via modulation of oxidative damage.

Besides oxidative stress, there is a decreased activity of glycolytic enzymes in the STZ model of memory deficit, which results in the reduction in acetylcholine level (Racchi et al. 2004; Ishrat et al. 2006), which is intricately associated with cognition. It is mainly found at the neuromuscular junctions and cholinergic neurons, where its activity serves to terminate synaptic transmission. Acetylcholine is required for the proper function of cholinergic transmission to regulate learning and memory deficit. Synthesis of AChE depends on the presence of acetyle-CoA (formed by the breaking down of glucose during glycolysis). It is degraded by the inhibitors of AChE, which are the most effective pharmacological approach for the symptomatic treatment of AD. The enhancement of cholinergic transmission by inhibition of AChE is the basis of the symptomatic treatment of dementia. In the present study, decreased AChE activity in the hippocampus leads to increased cholinergic transmission to facilitate the learning and memory deficit as documented by others (Sonkusare et al. 2005; Ishrat et al. 2006). ICV-STZ infusion has shown significantly increased AChE activity in the hippocampus, which is consistent with earlier studies (Racchi et al. 2004; Sonkusare et al. 2005; Ishrat et al. 2006). Moreover, ChAT plays a critical role by recycling acetylcholine neurotransmitters which are responsible for memory and cognition. ICV-STZ infusion has shown significantly increased AChE activity and decreased ChAT expression in the hippocampus, which is consistent with earlier studies (Racchi et al. 2004; Ishrat et al. 2006; Jin et al. 2009). WS pretreatment decreased the AChE activity and increased the ChAT expression by ameliorating oxidative loads in the hippocampus of STZ-infused rats.

Activation of caspase-3 is a key step in the execution process of apoptosis, and its inhibition can block apoptotic cell death. Elevated levels of oxidative loads are well capable of activating the apoptotic pathways (Marques et al. 2003) by exerting an excitotoxic effect on glutamate receptors via intracellular calcium influx, leading to apoptosis (Ghosh et al. 2011). On the other hand, free radical scavengers are known to improve cellular respiration in neurons undergoing apoptosis (Atlante et al. 1998; Zhang et al. 2011). A significant elevation in caspases-3 activity was noticed after STZ infusion in rats. However, increased caspase-3 activity markedly subsided in WS pretreated group and thus the anti-apoptotic protection afforded by WS may be attributed to its antioxidant and free radical scavenging potential as documented by other studies (Ahmad et al. 2005; Sehgal et al. 2012; Manjunath and Muralidhara 2012).

There are few limitations of this study. First, we have studied only the pretreatment effect of WS on STZ-induced cognitive impairment and neurodegeneration. Second, our study is acute that includes 21 days after ICV-STZ injection to the brain which is much smaller than the reported time (almost 6–9 months) to develop a pathological hallmark of Alzheimer’s disease like amyloid beta production and hyperphosphorylation of Tau. Further investigation into the therapeutic effect of WS on taupathy, beta-amyloid deposits, and cytoskeletal abnormalities is needed at different time points after ICV-STZ administration.

Conclusion

Our present investigations indicate that ICV-STZ cause behavioral deficits and oxidative stress due to free radical generation and downregulation of the antioxidant defense systems. WS offered significant neuroprotection in ICV-STZ-infused rats, which may attribute the improvement of behavioral deficit, inhibition of oxidative stress, apoptosis response, and upregulation of endogenous antioxidant status. Most of the neuroprotective effect afforded by the extract can be attributed to its main component Withaferin A, present in moderate quantity. The findings add to a growing body of research demonstrating the power of phytochemicals as broad-spectrum neuroprotective agents. Further investigation into the role and mechanisms of antioxidant action of WS is needed to determine whether it can be an effective remedy for neurodegenerative disease including AD.

Notes

Acknowledgments

The authors thank the Department of Ayurveda, Yoga and Naturopathy, Unani, Siddha, and Homoeopathy (AYUSH), Ministry of Health and Family Welfare, Government of India, New Delhi, for the financial assistance. The technical assistance of Dharamvir Singh and Abdul Fitr are greatly acknowledged. We greatly acknowledge Dr. Amrish Kumar Tiwari (M.V.Sc. Pathology), Jamia Hamdard, India for the histological interpretation for this manuscript.

Conflict of interest

The authors have no conflict of interest.

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Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Md. Ejaz Ahmed
    • 1
  • Hayate Javed
    • 1
  • Mohd. Moshahid Khan
    • 1
    • 4
  • Kumar Vaibhav
    • 1
  • Ajmal Ahmad
    • 1
    • 5
  • Andleeb Khan
    • 1
  • Rizwana Tabassum
    • 1
  • Farah Islam
    • 2
  • Mohammed M. Safhi
    • 3
  • Fakhrul Islam
    • 1
    • 3
  1. 1.Neurotoxicology Laboratory, Department of Medical Elementology and ToxicologyJamia Hamdard (Hamdard University)New DelhiIndia
  2. 2.Department of Biotechnology, Faculty of PharmacyJamia Hamdard (Hamdard University)New DelhiIndia
  3. 3.Neuroscience and Toxicology Unit, Faculty of PharmacyJazan UniversityJazanKingdom of Saudi Arabia
  4. 4.Department of Neurology, Carver College of MedicineUniversity of IowaIowaUSA
  5. 5.Department of Internal Medicine, Carver College of MedicineUniversity of IowaIowaUSA

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