Hippophae salicifolia D.Don berries attenuate cerebral ischemia reperfusion injury in a rat model of middle cerebral artery occlusion

Santhrani Thakur, Pradeepthi Chilikuri, Bindu Pulugurtha, Lavanya YaidikarInstitute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati-517502, Andhra Pradesh, India

Hippophae salicifolia D.Don berries attenuate cerebral ischemia reperfusion injury in a rat model of middle cerebral artery occlusion

Santhrani Thakur*, Pradeepthi Chilikuri, Bindu Pulugurtha, Lavanya Yaidikar
Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati-517502, Andhra Pradesh, India

ARTICLE INFO ABSTRACT

Article history:

Received 4 February 2015

Received in revised form 8 February 2015 Accepted 10 February 2015

Available online 11 February 2015

Keywords:

Hippophae salicifolia D.Don berries Ischemia reperfusion injury

Antioxidant

Anti-inflammatory

Anti-apoptotic activities

Objective: To investigate the protective effect of Hippophae salicifolia D.Don (H. salicifolia) berries extract against cerebral reperfusion injury induced neurobehavioral and neurochemical changes in a rat model of middle cerebral artery occlusion (MCAO). Methods: Rats were pretreated with alcoholic extract of H. salicifolia (250 and 500 mg/kg) for 14 d and focal cerebral ischemia was induced by MCAO. After 60 min of MCAO, reperfused for 24 h, a battery of behavioral tests were assessed the extent of neurological deficits. Infarct volume and brain edema were measured in 2,3,5-triphenyltetrazolium chloride stained brain sections. TNF-α, oxidative stress parameters like reduced glutathione, calcium, glutamate, malondialdehyde and apoptotic parameters like caspase-3, and caspase-9 were estimated in the brain homogenates. Results: Pretreatment with alcoholic extract of H. salicifolia at doses of 250 and 500 mg/kg significantly improved the neurobehavioral alterations and reduced the infarct volume, edema induced by ischemia reperfusion injury. H. salicifolia significantly prevented ischemia induced increase in malondialdehyde, glutamate, calcium, caspase-3, caspase-9 and TNF-αlevels as compared to ischemic animals. Conclusions: Our results indicate that H. salicifolia mitigated the ischemia reperfusion induced neuronal damage.

Tel: +91-9849077507

E-mail: drsanthrani@gmail.com

Foundation project: Supported by University Grants Commission (UGC. F. No. 39-174/2010 (SR), New Delhi.

1. Introduction

Stroke is considered as the third leading cause of death worldwide and the major cause of long-term disability in surviving victims. Most strokes are caused by focal occlusion of the cerebral blood vessel (ischemic stroke), which may be due to atherosclerosis or thrombosis and the reminders are the result of rupture of a blood vessel (hemorrhagic stroke)[1]. Ischemic stroke is the major form representing over 80% of the patients. Ischemic stroke is defined as brain cell death due to prolonged ischemia caused by occlusion of cerebral arteries, coupled with or without reperfusion. Reperfusion of occluded arteries by thrombolytic drugs within 3 h of onset of symptoms is the only approved pharmacological treatment for an ischemic stroke, the time constraint of which is hard to meet clinically. Thus, development of neuroprotectants that can complement reperfusion therapy are crucial, although most clinical trials testing neuroprotectants have failed to demonstrate any benefit. Under ischemic conditions, brain cells, especially neuronal cells are damaged by excitotoxicity in minutes and in hours by inflammation, oxidative stress and apoptosis. Neuronal death in a stroke is complex event involving failure of metabolic processes, excitotoxicity, loss of calcium homeostasis and oxidative stress[2]. The inflammatory response to brain injury plays a vital role in the pathogenesis of stroke[3,4], characterized by peripheral leukocyte infiltration and neurotoxicity of the cerebral parenchyma[5]. Infiltrated leukocytes exacerbate brain injury, whereas anti-leukocyte agents, adhesion molecule antagonists or cytokine inhibitors attenuate it[6,7], implied that agents with anti-inflammatory action could have therapeutic potential for the cerebrovascular disorder[8]. Neutrophilic infiltration in stroke is mediated by cytokines like TNF-α, IL-1β and IL-8. Oxidative stress, which results from an imbalance between the generationand removal of reactive oxygen species (ROS), probably plays an important role in the development of tissue damage induced by arterial occlusion with subsequent reperfusion[9,10]. Thus the neuroprotectants that inhibit excitotoxicity, inflammation, oxidative stress and apoptosis might be beneficial in attenuating cerebral infarction in ischemic stroke. Several neuroprotectants like pomegranate extract[11], s-allyl cysteine[12], solasodine[13], shinkonin[14], rutin[15], catechin[16], thymoquinone[17], silymarin[18], hesperidine[19], quercetin[20], curcumin[21], etc., showed protective effect against ischemia reperfusion injury.

In-depth survey of indigenous ethnobotanical knowledge of Hippophae salicifolia D.Don (H. salicifolia) revealed that the plant is traditionally utilized by local people of Central Himalaya in multidimensional aspects as food, fuel, medicine, veterinary care, cosmetics, agricultural tools and bio-fencing. The people of central Himalaya eat raw berries as a food. The fruit berries are utilized in the form of delicious chutney (local jelly), pickle, squash as well as juice. In northern Himalayan region, traditionally, H. salicifolia berries are used in microbial infections, pain, skin diseases, ulcers, malnutrition, liver disorders, jaundice, tumors and for promoting regeneration of tissues[22]. H. salicifolia berries are edible and nutritious. It has high content of vitamin C, tocopherols (vitamin E), dietary minerals, amino acids, carotenoids, flavonoidsisorhamnetin, quercetin, ω-3, ω-6 fatty acids, kaempferol, catechins, proanthocyanidins and chlorogenic acids[23].

The present study is designed to investigate the protective effect of H. salicifolia D.Don berries extract against cerebral reperfusion injury induced excitotoxicity, oxidative stress, inflammation and apoptosis in middle cerebral artery occlusion (MCAO) induced rats.

2. Materials and methods

2.1. H. salicifolia D.Don berries

H. salicifolia D.Don berries powder was purchased from Changsha Organic Herb Inc., China. The alcoholic extract was prepared by subjecting the berry powder to extraction with 90% alcohol, refluxed for about 6-8 h, filtered and evaporated to dryness by rotary film evaporator at low temperature under reduced pressure. The percentage yield was found to be 4.27% w/w. The extract was stored in dessicator and the weighed dose was suspended in 1% Tween-80 as and when required in the study.

2.2. Standardization of H. salicifolia

The alcoholic extract of H. salicifolia was subjected to preliminary phytochemical analysis[24,25]. The total phenolic content of the extract was determined with the Folin-Ciocalteau assay and expressed as milligrams of gallic acid equivalents (GAE) per 100 grams dry mass (mg GAE/100 g)[26]. The total flavonoid content was measured with an aluminium chloride colorimetric assay and expressed as milligrams of (+) quercetin equivalents (QE) per 100 gram dry mass (mg QE/100 g)[27]. The total tannin content was determined by Folin-Denis assay method and expressed as the equivalents of tannic acid (TE) (mg TE/100g extract)[28].

2.3. Chemicals

Trichloroacetic acid, 2-thiobarbituric acid, 5-51-dithiobis (2-nitrobenzoic acid), glutathione (GSH) and (±)-epinephrine were purchased from Sigma-Aldrich Co., Bangalore. 2,3,5-Triphenyltetrazolium chloride was purchased from Hi-Media, Mumbai. All other chemicals were of the highest purity commercially available. TNF-α ELISA kits were purchased from Ray Biotech, USA. Caspase -3, caspase -9 fluorometric kits were purchased from Calbiochem, USA.

2.4. Animals

Male Albino rats weighing 200-250 g were used. The animals were housed 5 per cage (440 mm×270 mm×178 mm) under controlled conditions of light (12h light/dark cycle, lights on at 7:00 a.m.), temperature [(22±2) °C] and humidity 50%-60% with free access to food and water. The animals were acclimatized to the laboratory for at least 7 d before they were tested. All the experiments were carried out between 9:00 a.m. and 3:00 p.m. after prior approval from Institutional Animal Ethical Committee No.1677/PO/a/12/ CPCSEA.

2.5. Acute oral toxicity studies

The acute toxicity studies were conducted as per the OECD guidelines 420 where the limit test dose of 2 000 mg/ kg used[29,30]. Observations were made for any mortality for about 24 h.

2.6. Experimental grouping and treatment

After acclimatization, the rats were randomly divided into four groups; each group consisted of 9 rats, treated with drug or vehicle orally once daily for 14 d prior to MCAO. The first group served as sham control and received 2% Tween 80 orally. The second group served as MCAO model group and received 2% Tween 80 orally. The third and fourth groups were treated with H. salicifolia 250 and 500 mg/kg p.o. respectively for 14 d. After 14 d of pretreatment, rats were subjected to 2 h right MCAO via the intraluminal filament technique and 22 h reperfusion. MCAO was performed according to previously described method[31,32]. Briefly, rats were anaesthetized with ketamine (100 mg/kg i.m.) and xylazine (10 mg/kg i.m.) the right common carotid artery was exposed at the level of the external and internal carotid artery bifurcation. A 4-0 nylon suture with a blunted tip was inserted into the internal carotid artery and advanced into the anterior cerebral artery to occlude the middle cerebral artery. After occluding the middle cerebral artery for 2 h, nylonsuture was removed carefully to restore blood flow and then the skin was sutured. Animals were then returned to their cages and closely monitored for 22 h. The body temperature was maintained at 37 °C with a thermostatically controlled infrared lamp. In sham-operated group, the external carotid artery was surgically prepared for insertion of the filament, but the filament was not inserted. After 22 h of reperfusion, neurological deficit was evaluated. Then six rats in each group were decapitated to obtain brain tissue samples for biochemical analysis, brain tissues of remaining rats in each group were used for the measurement of infarct volume.

2.7. Neurological deficit

After 24 h of the induction of ischemia-reperfusion, the animals were assessed for the presence of neurological deficits[31]. The scoring is as follows: 0: No apparent neurological deficit; 1: Failure to extend contra lateral forepaw fully; 2: Circling to contra lateral side; 3: Falling towards contra lateral side; and 4: Did not walk spontaneously and has a depressed level of consciousness.

2.8. Measurement of infarct volume

Rats were anaesthetized with ketamine (100 mg/kg i.m.) and xylazine (10 mg/kg i.m.), decapitated, brains were rapidly removed and cooled in saline at 4 °C for 15 min. Eight 2-mm thick coronal sections were then cut, beginning at the olfactory bulb. The slices were immersed in 2% 2, 3, 5-triphenyltetrazolium chloride solution, and kept at 37 °C in a water bath for 15 min. The slices were then digitally photographed. Unstained areas were defined as infarct, and were measured using image analysis software[33,34]. The infarct volume was calculated by measuring the unstained and stained areas in each hemisphere slice, multiplying this by slice thickness (2 mm), and then summing all of the eight slices. Corrected infarct volume=left hemisphere volume-(right hemisphere volume-infarct volume).

2.9. Brain tissue preparation for biochemical analysis of caspase-3, 9 and TNF-α

Brain samples were homogenized in ice-cold cell lysis buffer and kept at 40 °C for 1 h. Brain homogenates were centrifuged at 43 000 r/min for 15 min at 4 °C and supernatants were collected and stored at -80 °C until use. Protein contents were measured using the enhanced BCA protein assay kit.

2.9.1. Measurement of caspase-3 activity

Caspase-3 activity was measured in brain samples of the ischemic rats 24 h after reperfusion using a caspase-3/ CPP32 fluorometric assay kit (Calbiochem, USA). Cell lysates (20-200 μg protein) were incubated in 96-well plates with 2-fold reaction buffer (50 μL) and the reaction was started by the addition of 1 mmol/L fluorescent conjugated [allophycocyanin, 7-amido-4-trifluoromethyl chloride (AFC] caspase-3 Asp-Glu-Val-Asp peptide substrate (DEVDAFC) (5 μL) (Calbiochem, USA). After incubation in the dark at 37 °C, plates were read in a fluorimeter equipped with a 400 nm excitation filter and a 505 nm emission filter.

2.9.2. Measurement of caspase-9 activity

Caspase-9 activity was measured in brain samples from the ischemic rats 24 h after reperfusion using a caspase-9/ APAF-3 fluorometric assay kit (Calbiochem, USA). Cell lysates (20-200 μg protein) were incubated in 96-well plates with 2-fold reaction buffer (50 μL) and the reaction was started by the addition of 1 mmol/L fluorescent conjugated (allophycocyanin, AFC) caspase-9 fluorogenic substrate (LEHD-AFC) (5 μL) (Calciochem, USA). After incubation in the dark at 37 °C, plates were read in a fluorimeter equipped with a 400 nm excitation filter and a 505 nm emission filter.

2.9.3. Measurement of TNF-α

TNF-α was estimated from the brain homogenates by using rat TNF-α ELISA kit purchased from Raybiotech, USA.

2.10. Brain tissue preparation for biochemical analysis of antioxidant markers

The rats were sacrificed by cervical decapitation under anesthesia and brains were quickly dissected out, homogenized in 50 mmol/L phosphate buffer (pH 7.0) containing 0.1 mmol/L ethylene diamine tetraacetic acid to yield 5% (w/v) homogenate. The homogenate was centrifuged at 10 000 r/min for 10 min at 0 °C in cold centrifuge, the resulting supernatant was used for further studies.

2.10.1. Measurement of glutamate

Glutamate levels were measured according to the method described by Bernt and Bergmeyer[35], with minor modifications. To 1 mL of supernatant, 2 mL of perchloric acid was added and pH was adjusted to 9.0 with phosphate buffer. The resulting mixture was incubated at 37 °C for 30 min, subjected to centrifugation at 4 500 r/min for 15 min and was allowed to stand for 10 min in an ice bath and then filtered through fluted filter paper. Absorbance was measured at 340 nm. The glutamate levels are expressed as μmol/g tissue.

2.10.2. Measurement of calcium

A total of 0.5 mL of the sample was added to 4.5 mL of deproteinated buffer in a glass centrifuge tube and was placed in water bath for 3 min. Tubes were centrifuged while they were still hot, 0.5 mL of each supernatant was transferred into clean test tube. For the reagent blank, 0.5 mL of blank solution was prepared by mixing 9 volumes of deproteination buffer with one volume of water. Then, 5 mL of working coloring reagent was added to each tube, mixed well and then read at 570 nm[36].

2.10.3. Measurement of malondialdehyde (MDA)

MDA formation was estimated by the method of Ohkawa etal[37]. Briefly, 200 μL of brain homogenate supernatant was added to 50 μL of 8.1% sodium dodecyl sulphate, vortexed and incubated for 10 min at room temperature. A total of 375 μL of thiobarbituric acid (0.6%) was added and placed in a boiling water bath for 60 min and then the samples were allowed to cool to room temperature. A mixture of 1.25 mL of butanol: pyridine (ratio 1.5:1), was added, vortexed and centrifuged at 1 000 r/min for 5 min. The optical density of the colored layer was measured at 532 nm on a spectrophotometer against reference blank and the rate of MDA formed is expressed as nmol of MDA formed/h/mg protein.

2.10.4. Measurement of reduced GSH

GSH content was measured according to the method of Sedlak and Lindsay[38]. Briefly, 0.75 mL of brain homogenate supernatant was mixed with 0.75 mL of 4% sulphosalicylic acid and then centrifuged at 1 200 r/min for 5 min at 4 °C. From this 0.5 mL of supernatant was taken and added to 4.5 mL of 0.01 mol/L 5,5’-dithiobis-(2-nitrobenzoic acid), and the yellow color developed was read spectrophometrically at 412 nm immediately. The GSH content was calculated as nmol GSH/mg protein.

2.11. Statistical analysis

Data was expressed as mean±SEM and analyzed by One way ANOVA with post-hoc test of Dunnetts test using Graphpad Prism software (Version 5.0). A value of P＜0.05 was considered as statistically significant.

3. Results

3.1. Acute toxicity studies

Acute toxicity was studied by OECD guidelines 423 (limit test) at a dose of 2 000 mg/kg p.o. in female Albino mice. No mortality was observed at this dose. Hence, 5 000 mg/kg was considered as LD50cut off value. The dose selected was 250 and 500 mg/kg.

3.2. Preliminary phytochemical analysis and standardization of H. salicifolia

Preliminary phytochemical screening of the alcoholic extract of H. salicifolia berries revealed the presence of phenolic compounds, flavanoids, tannins. The total phenolic, tannin, flavonoid and vitamin content of alcoholic extract of H. salicifolia was found to be (1.24±0.15) mg/g GAE, (6.81± 0.96) mg/g TAE, (0.14±0.17) mg/g QE and (25.0±0.11) mg/g of ascorbic acid, respectively.

3.3. Effect of H. salicifolia on neurologic deficit

The neurologic score after 24 h of reperfusion is given in Figure 1. The rats pretreated with alcoholic extract of H. salicifolia showed reduced neurological score (P＜0.001) significantly as compared with ischemic control. Values reached normal in H. salicifolia higher dose pretreated rats.

Figure 1. Effect of H. salicifolia on neurological deficit score in rats subjected to 2 h ischemia and 22 h reperfusion.

*: P＜0.05, **: P＜0.01, ***: P＜0.001 vs sham control group; +++: P＜0.001 vs ischemic control group.

3.4. Effect on H. salicifolia on GSH levels

Ischemic control rats showed significantly decreased levels of reduced GSH (P＜0.001) as compared to sham control group ipsilaterally as depicted in Figure 2a. But there is no change in reduced GSH levels in all the groups contralaterally indicates the development of focal ischemia. Rats treated with higher dose of alcoholic extract of H. salicifolia showed significantly decreased (P＜0.001) GSH levels as compared to ischemic control group ipsilaterally.

3.5. Effect on H. salicifolia on calcium levels

Significant decreased levels of calcium is observed in ischemic rats (P＜0.001) as compared to sham control. Rats treated with alcoholic extract of H. salicifolia showed a significant reversal (P＜0.001) of MCAO induced increased calcium levels when compared to sham control ipsilaterally as depicted in Figure 2b. In H. salicifolia higher dose pretreated rats, calcium levels are restored to normal levels. But there is no change in calcium levels in all the groups contralaterally indicates the development of focal ischemia.

3.6. Effect on H. salicifolia on glutamate levels

The effect of alcoholic extract of H. salicifolia on glutamate levels in MCAO induced rats is showed in Figure 2c. MCAO induced ischemic control rats showed significantly increased glutamate levels (P＜0.001) when compared with sham control in ipsilateral brain section indicates the development of focal ischemia. But there is no change in glutamate levels in contralateral brain section. Rats treated with alcoholic extract of H. salicifolia showed significant attenuation in glutamate levels (P＜0.001) as compared to ischemic (MCAO) control. In H. salicifolia higher dose pretreated group, the values reached normal.

3.7. Effect on H. salicifolia on MDA levels

MDA levels were significantly increased (P＜0.001) inipsilateral brain sections of ischemic control rats when compared with sham control rats as shown in Figure 2d. But there is no change in MDA levels in contralateral brain section of all groups indicating development of focal ischemia in ipsilateral brain section of MCAO treated rats. Rats treated with alcoholic extract of H. salicifolia significantly restored MDA levels (P＜0.001) in ipsilateral brain sections and at higher doses values reached normal.

Figure 2. Effect of H. salicifolia on a: brain reduced GSH levels; b: on brain calcium levels; c: on brain glutamate levels; d: on brain MDA levels.Values are expressed as mean±SEM (n=6). Statistical difference was analyzed by One way ANOVA followed by post hoc Dunnetts test. *: P＜0.05 vs sham control group; **: P＜0.01 vs sham control group; ***: P＜0.001 vs sham control group; +++: P＜0.001 vs ischemic control group.

3.8. Effect on H. salicifolia on caspase-3 activity

Figure 3 shows the effect of H. salicifolia on caspase -3 activity in the brain experimental stroke induced rats. MCAO rats showed increased levels of caspase-3 activity which implies the development of focal ischemia. Alcoholic extract of H. salicifolia treatment significantly (P＜0.001) reversed caspase-3 activity as compared to ischemic (MCAO) rats in ipsilateral brain and the caspase-3 activity was restored to normal in H. salicifolia higher treatment group. But there is no change in all the groups of contralateral brain indicating development of focal ischemia.

Figure 3. Effect of H. salicifolia on brain caspase-3 activity.*: P＜0.05 vs sham control group; ***: P＜0.001 vs sham control group; +++: P＜0.001 vs ischemic control group.

3.9. Effect on H. salicifolia on caspase-9 activity

Figure 4 shows the effect of alcoholic extract of H. salicifolia on caspase-9 activity in MCAO induced rats. MCAO induced ischemic rats showed increased levels of caspase-9 activity. Groups pretreated with higher dose of alcoholic extract of H. salicifolia showed significant (P＜0.001), restoration in caspase-9 activity in brain ipsilaterally as compared to ischemic control group. But groups pretreated with lower dose of alcoholic extract of H. salicifolia did not show any significant change in caspase-9 activity as compared to ischemic control group, indicating lower dose is not sufficient to inhibit the caspase-9 activity.

Figure 4. Effect of H. salicifolia on brain caspase-9 activity.*: P＜ 0.05 vs sham control group; **: P＜0.01 vs sham control group; ***: P＜0.001 vs sham control group; +++: P＜0.001 vs ischemic control group.

3.10. Effect on H. salicifolia on TNF-αlevels

The effect of alcoholic extract of H. salicifolia on TNF-α levels in MCAO induced rats is shown in Figure 5. There is a significant increase (P＜0.001) in TNF-αlevels in ischemic control group as compared to sham control group in the brain ipsilaterally. But there is no change in all the groups of brain contra laterally. Groups pretreated with lower andhigher doses of H. salicifolia significantly (P＜0.01) restored ischemia induced increase in TNF-αlevels in brain ipsilaterally as compared to ischemic control group and the activity was restored to normal in H. salicifolia higher treatment group.

Figure 5. Effect of H. salicifolia on brain the TNF-αlevels.**: P＜0.01 vs sham control group; ***: P＜0.001 vs sham control group; +++: P＜0.001 vs ischemic control group.

3.11. Effect on H. salicifolia on infarct volume

The results of the effect of H. salicifolia on brain infarct volume in experimental stroke induced rats is summarized in Figures 6 and 7. Alcoholic extract of H. salicifolia showed a significant reversal in ischemia induced increased brain infarct volume (P＜0.001) as compared to ischemic control group and the values reached normal in H. salicifolia higher treatment group.

Figure 6. Effect of H. salicifolia on infarct area.*: P＜0.05 vs sham control group; ***: P＜0.001 vs sham control group; +++: P＜0.001 vs ischemic control group.

Figure 7. The 5 coronal sections of rat brain with 2,3,5-triphenyltetrazolium chloride staining, in the sham and ischemic control and alcoholic extract of H. salicifolia treated groups.

Red and colourless denote the normal and infarcted areas, respectively. The colourless region corresponds to occluded middle cerebral artery territory.

4. Discussion

The study was aimed to examine whether treatment with H. salicifolia D. Don berries extract has neuroprotective effect in experimental stroke induced rats. The mechanism of stroke is still unclear. So far, it is believed that the mechanism consists of excitotoxicity, free radical injury, apoptosis, inflammatory response and so on[39].

The MCAO model is by far the most commonly used stroke model, since the pathophysiology presented in this model closely matches that in stroke patients, many of those patients suffered from focal cerebral arterial thrombosis[31].

Neurological test is a common method to estimate the success rate of MCAO modeling and the degree of ischemia. In our study, the neurological test showed the curative efficacy of H. salicifolia on ischemia injury. We employed most commonly used neurological test to assess the altered function of brain following focal cerebral ischemia in rats. In rats, occlusion of the middle cerebral artery resulted in significant neurological deficit, which was evident from the neurological score[40]. According to our observations, H. salicifolia higher dose treatment (500 mg/kg) showed significantly decreased neurological deficit scores. These changes were also reflected in the infarct volume, which is the most vigorous index of ischemic brain damage. In the present study all the antioxidant parameters are measured in ipsilateral (on the same side of ischemic brain) and contralateral (on the opposite side of ischemic brain) section of brain to confirm the induction of focal ischemia and also to confirm whether biochemical changes occurs locally or induce changes on the opposite side of the brain as well.

Free radicals have been implicated in cerebral ischemia and reperfusion-induced neuronal injury[41,42]. Free radicals promote lipid peroxidation which results in the alteration in permeability and fluidity of membrane[43]. ROS produce MDA, an end product of lipid peroxidation. MDA estimation correlates with ROS formation. In the present investigation pre-treatment with berries extract of H. salicifolia reversed focal ischemia and reperfusion-induced increase in the level of MDA as compared to sham control group.

A number of processes have been implicated in the pathogenesis of ischemia-induced cell death. The first consequence is cerebral blood flow reduction resulting in depletion of substrates, particularly oxygen and glucose that cause energy failure leading to perturbation of the membrane bound ion-pumps (like Na+-K+ATPase) leading to ion dyshomeostatis (elevates intracellular Na+, Ca2+, Cl-and extracellular K+levels). In healthy conditions, only α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptors are activated by low concentrations of glutamate. At this low glutamate concentration, N-methyl-D-aspartate (NMDA) receptor channels remain blocked by Mg2+ions. This low concentration of glutamate is insufficient to open metabotrophic receptor. In the very early phase of ischemia,membrane potential is lost due to depletion of presynaptic neuronal energy and glia depolarize to release large amounts of excitotoxic amino acids (especially glutamate). However, during ischemia, repeated action of glutamate at AMPA receptors cause sustained post synaptic depolarization, unblocks inhibitory control of Mg2+ion at NMDA receptor which allows entry of Ca2+and Na+ions[44]. Accumulated Ca2+ions further releases glutamate. The released glutamate load activates metabotrophic receptor also, which further releases Ca2+ions from endoplasmic reticulum through IP3cascade[45]. Glutamate release is proportional to the amount of Ca2+accumulation, activation of NMDA and metabotrophic receptor, a vicious cycle of Ca2+and glutamate release is formed. Ca2+overload results in failing of normal feed back mechanism, leading to dangerous excitotoxicity, which activates protein kinase-C[46,47]. AMPA receptor channel is phosphorylated by protein kinase-C (PKC) results in persistant depolarization and recruitment of more AMPA receptors. Further, PKC activates lipases and neuronal nitric oxide synthase (nNOS). nNOS converts L-arginine to nitric oxide (NO), which further facilitates glutamate release and the sequence of receptor activation. PKC phosphorylates signal transduction molecule controlling gene transcription in the post synaptic cells leading to permanent increase in the number of synaptic clefts. Membrane damage is aggravated by lipases, which form lipid peroxides. In addition, NO in presence of ROS, generate peroxynitrite and hydroxyl free radicals, which independently cause neurotoxicity. Severe neuronal damage releases polyamines and arachidonic acid which potentiate excitotoxicity.

Activation of NMDA receptors and accumulation of Ca2+stimulate the synthesis of transcription factors such as nuclear factor-kB and interferon regulatory factor 1 which regulates the inflammatory cytokine production. TNF-αis a pivotal central regulator of inflammation and has potent stimulatory effects in immune and vascular response, increases rapidly in the brain lesion tissue after experimental brain ischemia[48]. The TNF-α causes cerebral edema by increasing leucocyte and potent vasoactive agents of endothelin-1 and nitric oxide invasion to the ischemic area which further increase brain damage. TNF-α inhibits the reuptake of glutamate leads to over activation, upshots in increased Ca2+levels and cerebral edema. It was found that increased cytokine levels were associated with increased Ca2+levels and infarct size, neurological history[49].

Cytoplasmic calcium activates proteolytic enzymes and second messenger cascades that contribute to cell death. Activated proteolytic enzymes break down elements of the cytoskeleton, leading to protein aggregation. Calciummediated lipolysis damages membranes and forms lipid peroxides in presence of free radicals. Apoptotic cascades are stimulated by the rise in calcium through mitochondrial permeability. In the present study results demonstrated that calcium levels were elevated in Ischemic control group compared to normal group rats which is in consonance with the earlier reports[44]. A significant decrease in total calcium level was observed in H. salicifolia pretreatment groups. Overproduced free radicals are detoxified by endogenous antioxidants. Glutathione is considered a central component in the antioxidant defense of cells. It acts both to directly detoxify ROS and as a substrate for various peroxidases[50]. Pre-treatment with berries extract of H. salicifolia significantly prevented focal cerebral ischemia induced decline in GSH content and increase in MDA, glutamate and TNF-α levels indicating atleast inpart the calcium lowering activity may be due to decreased levels of glutamate, TNF-αor antioxidant capacity of H. salicifolia.

Several lines of evidence argue that neuronal loss during the reperfusion is the result of activation of celldeath programs including apoptosis[51,52]. Two pathways of apoptosis have been identified, the ‘extrinsic’ and ‘intrinsic’pathway. Induction of the extrinsic pathway of apoptosis is associated with the activation of extracellular TNF super family cell-death receptors; these then recruit other proteins to form a complex that activates caspase-8, and this in turn activates caspase-3 (executioner caspase). In the intrinsic pathway, mitochondrial damage activates caspase-8, which activates caspase-3. This ‘executioner’ caspase has multiple cell-death effects including the induction of widespread proteolytic activity, DNA breakdown, inhibition of DNA-repair enzymes, and disruption of the cytoskeleton. A number of in vivo studies have demonstrated recently the activation of caspases like caspase-3, caspase-8 and caspase-9 during cerebral ischemia and infarction[53-59]. Furthermore, caspase-3 and caspase-9 deficient mice were found to be more resistant to ischemic stress and showed a significant reduction in stroke volume[60]. Together, these findings stimulated interest in the development of specific caspase inhibitors as therapeutic agents in cerebral ischemia[61]. Ischemic group showed increased TNF-α and caspase-9 indicating the involvement of extrinsic and intrinsic pathways. HS at 500 mg/kg treatment reversed ischemia induced increase in caspase 3, caspase-9 and TNF-αlevels as compared to ischemic control group ipsilaterally indicating its activity on extrinsic and intrinsic apoptotic pathway. The anti-apoptotic properties of HS were proven by reversing increased caspase-3, 9 and TNF-α activity in brain cells.

From our results, it can be concluded that the H. salicifolia D.Don berries extract attenuates cerebral reperfusion injury induced oxidative stress, apoptosis and inflammation in a rat model of stroke, and neuroprotection is likely related to attenuation of oxidative stress, apoptotic mediators like caspase-3, caspase-9 and inflammatory mediator like TNF-α, leading to inhibition of apoptotic cell death.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgements

The authors would like to thank University Grants Commission for their financial grant support [UGC. F. No. 39-174/2010 (SR), New Delhi] to carry out this research work.

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*Corresponding author:Santhrani Thakur, Department of Pharmacology, Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam, Tirupati, Andhra Pradesh, India.