1. INTRODUCTION:

A stroke is state of hypoxia due to diminution of cerebral blood flow (CBF) to a critical threshold that propagates brain damage involving the entire brain or a selective region by a clot in vessel or artery or breaking of blood vessel.^[^1]According to American heart association, in 2015, there will be 795,000 people diagnosed of stroke and in the same year 129,000 cases (about 11.13%) of death were reported. Stroke was the second commonest cause of mortality worldwide and the third commonest cause of mortality in developed countries. Stroke is the fourth commonest cause of death in the United States. Stroke hitting rate in US, is estimated as one stroke in every 40 seconds.^[^2]

Hypertension may be major risk factor for stroke. A high intraluminal pressure will lead to extensive alteration in endothelium and smooth muscle function in intracerebral arteries. Which can increase permeability over the blood-brain barrier and lead to local or multifocal oedema of brain. Altered endothelium and blood cell-endothelium interaction can lead to local thrombi formation and ischaemic lesions.^[^3]

Neurons are particularly susceptible to ischaemic injury because they have limited energy stores and higher energy demand. Stored energy of intrinsic central nervous system deplete within 2 to 4 minutes of anoxia. Which lead to anaerobic glycolysis and then lactic acidosis. Energy failure disrupts ion homeostasis. Cellular influx of sodium & chloride with osmotically obligated water and the influx of calcium occur.^[^4] Calcium influx may be responsible for activation of phospholipase C with breakdown of phospholipids in the cell membrane and liberation of free fatty acids. Which is responsible for direct tissue damage.^[5] When brain cells begin to die, lead to abilities controlled by that area of the brain are lost including functions such as speech, movement, and memory.^[4]

In ischemic condition oxygen supply is limited, leads to the electron transport chain of the inner mitochondrial membrane highly reduced, result in oxygen radical production.^[5]Reperfusion can restore cerebral blood flow (CBF), but can lead to secondary brain injury from influx of neutrophils, generation of reactive oxygen species (ROS), hemorrhage and cerebral edema.^[1,5]

Present therapy for ischemic injury involves two distinct strategy i.e. neuroprotection and vascular reperfusion. For this purpose we selected two well estabilished antihypertensive drugs to evaluate preventive neuroprotective effect. A wide variety of neuroprotectives have been studied in animal models but they were found to be failed in clinical trial. At present only rt-PA (reverse transcriptase tissue plasminogen activactor) is available for the treatment of cerebral ischemic injury, which act through vascular reperfusion.^[^6]

Amlodipine is an orally active long lasting dihydropyridine calcium channel blocker (CCB) approved for the treatment of hypertension, angina, and coronary artery disease. Calcium channel blockers might ameliorate the effect of cerebral ischemia by mechanism as: relaxation of vascular smooth muscle, resulting in vasodilation and improved cerebral perfusion and prevention of calcium flux into neurons, thereby limiting activation of phospholipases, proteases, and consequent membrane and protein degradation and production of damaging metabolic by-products such as free radicals.^[7,8] Steady state plasma levels of Amlodipine are reached after 7 to 8 days of consecutive daily dosing.^[9]

Telmisartan is a Angiotensin II type 1 receptor (AT1 Receptor) blocker and is a unique drug that has a neuroprotective action and acts as an agonistic ligand for peroxisome proliferator-activated receptor-gamma (PPARγ).^[10] Telmisartan also shows anti-inflammatory effect.^[11] Telmisartan is indicated in the treatment of essential hypertension and the prevention of CV events. Steady state plasma concentrations of Telmisartan is achieved within 7 days of consecutive daily dosing.^[^12]

High doses of Calcium channel blocker may responsible for periferal edema. But in combination with AT1 receptor blocker (Telmisartan) low dose of calcium channel blocker is sufficient. Calcium channel blocker cause pre-capillary vasodilatation and then increase capillary hydrostatic pressure, Telmisartan can normalize transcapillary pressure and reduce oedema by postcapillary vasodilatation.^[^13]

Aim of this research was to find out possible use of well known antihypertensive drugs at their therapeutic doses, in prevention of ischemia and reperfusion induced cerebral injury.

2. MATERIALS AND METHODS:

Drugs:

Amlodipine tablet [USV Ltd., Baddi, India] (B.N. Amlopin-5, B.No. 48001347),

Telmisartan tablet [Dr. Reddy Lab., Hyderabad, India] (B.N. Telsartan 20, B.No. TA20603).

Animals:

Male wistar rats having weight 200-250 gm were used. Rats were obtained from animal house of SKNCOP, Pune. All the rats were maintained in colony cages under controlled conditions of temperature (28 ºC), light (10 h light: 14 h dark), humidity (50 F, 5percent) and permitted ad libitum access to standard lab chow and tap water before and after experimental procedures.^[^14] All the procedures were performed according to CPCSEA guidelines with prior approval of Institutional Animal Ethics Committee (IAEC) for experiment on animals (Protocol no. IAEC-54-10-2012).

Section groups and drug treatment:

The animals were randomly divided into five weight-matched groups (n= 6).

Group I: Sham group (n=6); received normal saline (10 ml/kg, once a day), No ischemia only surgical exposure.^[^15]

Group II: control group (n=6); received normal saline (10 ml/kg, once a day), 10 min occlusion.^[^16]

Group III: Amlodipine treated group (n=6); received Amlodipine (1mg/kg, once a day), 10 min occlusion^[^17]

Group IV: Telmisartan treated group (n=6); received Telmisartan (6 mg/kg, once a day), 10 min occlusion^[^17]

Group V: Combination (Amlodipine+Telmisartan) treated group (n=6); received combination of (Amlodipine 1mg/kg + Telmisartan 6 mg/kg, once a day), 10 min occlusion,.^[^17]

Normal saline and all the drugs were administered orally for seven days till before BCCAO (bilateral common carotid artery occlusion) to their respective groups.^[^18]

Induction of ischemia:

Chloral hydrate (350 mg/kg, i.p.) was used as anesthetic agent.^[^18] Both common carotid arteries were exposed over a midline incision, and a dissection was made between the sternocleidomastoid and the sternohyoid muscles parallel to the trachea. Each carotid artery was freed from its adventitial sheath and vagus nerve, which was carefully separated and maintained. The induction of ischemia was performed by occluding bilateral common carotid arteries (BCCA) with clamps for 10 min^[^19] followed by 24 h reperfusion^[20] and the skin was closed with stitches using waxed silk suture. Sham animals received same surgery, expose both carotid artery without occlusion.^[^14] After the

operation, the animals were then returned to their cages for 24 hour reperfusion period with free access to food and water.^[18]

Neurobehavioural parameters

a) Spatial memory evaluation (by Elevated plus maze apparatus):^[21]

Spatial memory was evaluated by using elevated plus-maze apparatus (Kulkarni, 2007).^[^22] In this experiment, individual rat was placed at the end of the open arm facing away from central platform of the maze. The time taken by the rat to enter from open arm with all the four legs into the enclosed arm was taken as transfer latency time (TLT). In case the animal did not enter the enclosed arm within 90 sec, it was gently pushed and a TLT of 90 sec was assigned to it. Acquisition of memory was assessed on 6th day before the occlusion, and that of retrieval of memory after 24 hr reperfusion (8^th day).^[^21]

b) Locomotor activity (by Actophotometer):

The locomotor activity (ambulatory activity) was recorded by using actophotometer [INCO, India]. Animal was placed individually in the Actophotometer for 3 min for habituation and thereafter, locomotor activity was recorded by placing the animal for 5 min in Actophotometer. Ambulatory activity was expressed in terms of total photo beam counts within 5 min.^[^15,18]

c) Balance and motor Coordination and (by Rotarod apparatus):

Rotarod apparatus [INCO, India] is used to assess balance and motor coordination by observing retention time of rats on revolving rod (Dunham and Miya, 1957).^[^23,24] The Rota-rod cylinder was accelerated from 4 to 40 rpm within five minutes. Animals existed and performed on revolving rod were for more than 160 seconds were included in the study and the cutoff time was fixed for 300 seconds.^[^25]

Biochemical parameters:

After 10 min occlusion and 24 hour reperfusion, animals were sacrificed by decapitation and brain of each animal was removed (within 60 seconds)^[26] and washed in ice cold saline (0.9percent w/v).^[21] It was homogenized in 10 percent w/v of ice cold phosphate buffer saline (0.1 M, pH 7.4) by teflon glass homogenizer. The homogenate was centrifuged at 1000× g at 4 ◦C for 3 min and the supernatant divided into two portions, one of which was used for measurement of lipid peroxidation (LPO) assay.^[^27] The remaining supernatant was again centrifuged at 12,000× g at 4 ◦C for 15 min and used for Super oxide (SOD) assay. Post mitochondrial supernatant (PMS) was kept at −10 ◦C until assayed.^[^28,29,30]

a) Lipid peroxidation:

Lipidperoxidation is estimated in form of Malonaldehyde, which is obtained as an end-product of oxidation of polyunsaturated fatty acids. In lipid peroxidation assay, the Melonaldehyde react with thiobarbituric acid (TBA) to form a pinkish-red chromagen, which can be estimated at 532nm absorbance. (Ohkawa et al., 1978).^[^31]

Briefly, to 0.2ml of brain homogenate, 8.1 % w/v sodium dodecyl sulphate, 20 % v/v acetic acid (pH 3.5), and 0.8 % w/v aqueous TBA were added and heated in an oil bath at 95ºC for 60min. After cooling, mixture of n-butanol and pyridine (15:1 v/v) were added and centrifuged at 4000 rpm for 10min. absorbance of organic pink layer was measured at 532nm.^[32] 1,1,3,3-tetramethoxy propane was used as an external standard, and the level of lipid peroxidation was expressed as nmol/mg of MDA.^[33]

b) SOD (Superoxide dismutase) activity:

SOD activity can be expressed as the capacity of tissue SOD to inhibit the auto-oxidation of epinephrine to adrenochrome at alkaline pH (Misra and Fridovich, 1972).^[^34]

Briefly, to 0.5 mL of brain tissue homogenate, ice cold distilled water, ethanol, chloroform were added and centrifuged at 2500 rpm at 4°C for 15 minutes. To 0.5 mL of supernatant, EDTA (0.3 mM) and carbonate-bicarbonate buffer (0.05 M) pH 10.2 were added. The reaction was initiated by the addition of substrate (Epinephrine 0.3 mM) and the increase in absorbance was recorded at 480 nm.^[^35,^36] The enzyme unit of activity was defined as, the enzyme required for 50 percent inhibition of epinephrine auto oxidation.^[37]

Statistical analysis:

Data is presented as mean± SEM. Data of MDA assay, SOD activity and locomotor activity were analysed by one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Data of spatial memory were analysed by repeated measure ANOVA followed by Tukey’s multiple comparison test. And data of rotarod test were analysed by one way ANOVA followed by Bonferroni post-tests. A p-value of (^*P <0.05) was considered statically significant.^[^18,24]

3. RESULTS:

a) Effect on spatial memory: Spatial memory is determined by percentage change in transfer latency time. After 10 min occlusion and 24 hr reperfusion, transfer latency time (TLT) was increased non significantly in control group as compare to sham group. Telmisartan alone and in combination with Amlodipine showed percent decrease (non significantly) in Transfer latency time (TLT) and which is nearly same as sham group. Amlodipine showed slight percent increase in TLT compare to sham group. (Figure 1).

No marked difference was seen in retention time post reperfusion compared to pre occlusion in all groups, except Telmisartan group which showed decrease in retention. (Figure 3). All values are expressed as mean±SEM. Data was analysed by One way ANOVA followed by Bonferroni post tests. ns (non significant) as compared to preocclusion by using graph pad prism 5. N= 6 animals.

d) Effect on lipid peroxidation: Lipid peroxidation is determined as MDA content in tissue. After 10 min bilateral common carotid artery occlusion and 24 hour reperfusion, lipid peroxidation was increased nonsignificantly in control group as compared to sham group. Amlodipine alone and in combination with Telmisartan showed nonsignificantly decrease in lipid perixidation in brain tissue as compared to control group but Telmisartan showed slight increase in lipid peroxidation as compared to control group. (Figure 4).

Figure 4: Effect of drugs treatment on lipid peroxidation.

MDA content was increased nonsignificantly in control group as compared to sham group. Amlodipine and combination group showed nonsignificantly decrease in MDA content in brain tissue as compared to control group. MDA content of Telmisartan and control group was same. (Figure 4). All values are expressed as mean±SEM. Data was analysed by One way ANOVA followed by Tukey’s multiple comparison test. ns (non significant) as compared to sham group and control group by using graphpad prism 5. N= 6 animals.

e) Effect on Superoxide dismutase (SOD) activity: SOD activity is shown by percent inhibition of Adrenaline oxidation by SOD enzyme. After 10 min cerebral ischemia and 24 hr reperfusion, SOD activity was decreased nonsignificantly in control group as compared to sham group. All drug treated groups showed significantly increase in SOD activity as compared to control group. (Figure 5)

Figure 5: Effect of drugs treatment on SOD activity.

SOD activity was decreased nonsignificantly in control group as compared to sham group. All drug treated groups showed significantly increase in SOD activity as compared to control group. All values are expressed as mean±SEM. Data was analysed by One way ANOVA followed by Tukey’s multiple comparison test. ns (non significant) as compared to sham group and ^*P<0.05 as compared to control group by using graphpad prism 5. N= 6 animals. SOD (Superoxide Dismutase).

4. DISCUSSION:

Hypertention is major cause of brain stroke (cerebral ischemia).^[^2] So experimental objective was to find out such antihypertensive drugs which may have preventive neuprotective efficacy. That drugs should be lipophilic to cross blood brain barrier. For this purpose we selected combination of Amlodipine (1mg/kg) and Telmisartan (6 mg/kg), because this combination has been shown to exhibit synergistic antihypertensive effect in rat model.^[^17] Moreover, high dose Amlodipine may cause pereferal edema, because of increased capillary hydrostatic pressure from pre-capillary vasodilatation, while AT I antagonists (Telmisartan) can cause postcapillary vasodilatation, result in normalizing transcapillary pressure and reducing edema.^[13]

Calcium channel blockers can produce vasodilatory effect and calcium flux blocking effect into neurone, to prevent ischemia and generation of free redicals.^[^7]Telmisartan shows vasodilatory and anti-inflammatory effect.^[10,
11] Telmisartan abrogates pro-inflammatory effects of IL-1β by mechanisms involving inhibition of NADPH oxidase activation and the JNK/c-Jun pathway.^[11] Moreover, angiotensin II and AT1 receptors are also found in brain.^[38]

Poststroke memory dysfunction, has been proved in previous studies.^[^39] In present study, spatial memory was affected in control group as compared to sham group. But all drugs treated groups (Amlodipine, Telmisartan, Combination) maintain spatial memory after stoke as compared to control group (Figure 1). It is suggested that Amlodipine, Telmisartan and combination (Amlo.+Telmi.) may prevent neurones from ischemic injury. As discussed previously, theoretically calcium channel blocker Amlodipine can prevent depolarization induced neuronal injury. But Telmisartan can target angiotensin II receptors directly in brain. According to previous studies angiotensin II increases the production of reactive oxygen species (ROS) in cerebral microvessels via gp91phox (nox-2), a subunit of NADPH oxidase. Continuous stimulation with angiotensin II may damage neurons via multiple cascades through AT1 receptor stimulation. While, unopposed AT2 receptor is expected to prevent neural damage and cognitive impairment. Stimulation of the AT2 receptor may promote cell differentiation and regeneration in neuronal tissue and also support neuronal survival and neurite outgrowth against ischemic injury. AT2 receptor signaling can repair damaged DNA through induction of a neural differentiating factor, methyl methanesulfonate-sensitive-₂ (MMS₂).^[^40] So Telmisartan alone or with combination might prevent or repair neuronal injury.

Previous studies has proved the effect of stroke on walking ability, as in one study, only 37% of stroke survivors were able to walk after one week of stroke.^[41] These results support the results of our study, where diminished locomotor activity was found in control group as compared to sham group. While, all drugs treated groups including Amlodipine, Telmisartan and Combination (Amlo.+Telmi.) groups, showed enhanced locomotor activity compare to control groups and sham group, (Figure 2) Suggest neuropreventive ability of Amlodipine, Telmisartan and Combination (Amlo.+Telmi.).

In previous clinical studies, MRI analysis of stroke patients with impaired motor function, revealed that lesions were more common in paravermal lobules IV/V and affected the deep cerebellar nuclei.^[42] Another preclinical study demonstrated that after 24 hr of stroke, balance and motor coordination was unchanged as compared to preocclusion state, and that was affected after seven days of stroke.^[25] Our results of post occlusion balance and motor coordination gave so complex to make any inference. When we compared post occlusion results with pre occlusion results, we found pre and post occlusion results were nearly same, after 24 hour of stroke, no significant changes in balance and motor coordination are found compare to preocclusion. (Figure 3).

Malonaldehyde (MDA) is the end product of lipid peroxidation. Reactive oxygen species (ROS) and nitrogen species (RNS) are increased during ischemia and reperfusion. These free redicals can cause lipid peroxidation, membrane injury, disrupt cellular processes, and DNA damage.^[^44] In our study, after stroke lipid peroxidation was increased in control group as compared to sham group. Amlodipine group and combination group showed, decreased lipid peroxidation as compared to control group. But Telmisartan group increased MDA content unexpectedly.It can be correlated it with eNOS pathway. Where, Telmisartan inhibits vasoconstriction through increased nitric oxide (NO) production arising from the elevated eNOS expression and phosphorylation via a PPARγ-dependent mechanism.^[^43] In oxidative stress condition there is formation of reactive nitrogen species (RNS) and hydroxyl redical like reactive species instead of NO formation. Which lead to neuronal damage.^[^{15, 43]} This study has showed preventive effect of Amlodipine and its combination with Telmisartan in neuronal injury. (Figure 4)

Superoxide anion is an important factor for the development of infarction by brain edema formation and apoptotic neuronal cell death after focal cerebral ischemia and reperfusion.^[^45] Superoxide dismutase (SOD) is a bio enzyme, which dismutase the metabolism of superoxide ions into H₂O₂ and O₂. Principle of SOD activity estimation is that percent inhibition of adrenaline auto oxidation into adrenochrome, which give absorbance at 480nm.^[34] In present study, after stroke SOD activity was decrease as compared to sham group. All drug treated groups including Amlodipine, Telmisartan and combination (Amlodipine+Telmisartan) showed significant increase in SOD activity. (Figure 5). Suggest significant neuropreventive effect of Amlodipine and Telmisartan and their combination.

5. CONCLUSION:

Seven days pre-treatment with therapeutic doses of combination of Amlodipine (1 mg/kg) and Telmisartan (6 mg/kg) has been shown beneficial effect against ischemic stroke and neuronal damage compared to control group treated only with normal saline. The protecting effect of the combination may be due to combined antioxidant effect as alteration in In-vivo antioxidant has been observed. So the combination may be useful in reducing incidence of stroke in hypertensive patients. More studies with different parameters are required in other experimental models and cell lines to confirm the neuroprotective activity of commonly used antihypertensive drugs.

6. LIST OF SYMBOLS:

ºF	Degree fahrenheit
G	Gram
Gm	Gram
H	Hour
Hr	Hour
i.p.	Intraperitonial route
M	Molar
mg/kg	Milligram per kilogram
Ml	Mili litter
ml/kg	Mili litter per kilogram
mM	Mili molelar
N	Number
Nm	Nano meter
nmol/mg	Nano mol per mili gram
Ns	Non significant
^oC	Degree centigrade
pH	pH is a measure of the acidity or basicity of an aqueous solution.
v/v	Volume per volume
w/v	Weight per volume

7. LIST OF ABBREVIATIONS:

ANOVA	Analysis of Variance
AT	Angiotensin
B.N	Brand name
B.No.	Batch number
BCCA	Bilateral common carotid artery
BCCAO	Bilateral commom carotid artery occlusion
CBF	Cerebral blood flow
CCB	Calcium channel blocker
CPCSEA	Committee for the Purpose of Control and Supervision of Experiments on Animals
DNA	Deoxyribonucleic acid
EDTA	Ethylene diamine tetra acetic acid
eNOS	endothelial nitric oxide synthase
GPD	Glyceraldehydes-3-phosphate dehydrogenase
IAEC	Institutional Animal Ethics Committee
IL-1β	Interleukin-1 beta
JNK-c	c-Jun N-terminal kinases
LPO	Lipid peroxidation
MDA	Malonaldehyde
MMS₂	Methyl methanesulfonate-sensitive₂
NADPH	Nicotinamide adenine dinucleotide phosphate
NO	Nitric Oxide
PG	Prostaglandins
PMS	Post mitochondrial supernant
PPARγ	Peroxisome proliferator-activated receptor
RNS	Reactive Nitrogen Species
ROS	Reactive Oxigen Species
RPM	Revolutions per minute
rt-PA	Reverse transcriptase tissue activactor
SEM	Standard error of mean
SOD	Superoxide dismutase
TBA	Thiobarbituric acid
TLT	Transfer Latency Time
WHO	World Health Organization

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Received on 12.03.2015 Modified on 20.03.2015

Res. J. Pharmacology & P’dynamics. 7(1): Jan.-Mar. 2015; Page 11-18

DOI: 10.5958/2321-5836.2015.00003.8

Intraperitonial route

Ethylene diamine tetra acetic acid

Revolutions per minute