effect of ethanolic extract of root of pongamia pinnata (l...

Indian Journal of Experimental Biology Vol. 45, October 2007, pp. 868-876

Effect of ethanolic extract of root of Pongamia pinnata (L) pierre on oxidative stress, behavioral and histopathological alterations induced by cerebral ischemia–

reperfusion and long-term hypoperfusion in rats

Raghavendra Ma, Anshuman Trigunayata, R K Singhc, S Mitrab, R K Goela & S B Acharyaa* aNeuropsychopharmacology Laboratory, Department of Pharmacology, bDepartment of Pathology, Institute of Medical

Sciences, Banaras Hindu University, Varanasi 221005, India cCentral Research Institute (Ayurveda), Salt Lake, Kolkata, 700091, India

Received 25 August 2006; revised 3 July 2007

Possible effect of an ethanolic root extract of Pongamia pinnata (L) Pierre (P. pinnata) on oxidant-antioxidant status and histopathological changes in acute ischemia-reperfusion injury in the rat forebrain have been investigated. Further, its effect was also assessed on long-term cerebral hypoperfusion-induced changes in anxiety, cognitive and histopathological parameters. Cerebral post-ischemic reperfusion is known to be associated with generation of free radicals. In the present study, bilateral common carotid artery occlusion (BCCAO) for 30 min followed by 45 min reperfusion produced increases in lipid peroxidation, superoxide dismutase (SOD) activity and a fall in the total tissue sulfhydryl (T-SH) levels. The ethanolic extract of roots of P. pinnata (50 mg kg-1, po for 5 days) attenuated the ischemia-reperfusion-induced increase in lipid peroxidation, SOD activity and a fall in T-SH levels. The extract also ameliorated histopathological changes and inflammatory cell infiltration in the frontoparietal region of the rat brain. The extract (50 mg kg-1, po for 15 days) was also found to alleviate the long-term hypoperfusion-induced anxiety and listlessness (open field paradigm). There was an improvement of learning and memory deficits (Morris’ water maze testing). It also attenuated reactive changes in forebrain histology like gliosis, lymphocytic infiltration, astrocytosis and cellular edema. Results suggest protective role of P. pinnata in ischemia-reperfusion injury and cerebrovascular insufficiency states.

Keywords: Behavior, Cerebral hypoperfusion, Cognition, Oxidative stress, Pongamia pinnata, Reperfusion injury

The plant, Pongamia pinnata (L) Pierre (Leguminosae) (Synonym Pongamia glabra Vent.) (Karanj in Hindi) is widely used in several Ayurvedic medicinal formulations for its diverse medicinal properties1,2. The ethanolic extract of P. pinnata roots is reported to have antiinflammatory, gastroprotective and antistress activity3,4. It is known to have antioxidant property5 and has also been reported to reverse alterations in central cholinergic markers of cognition in experimentally demented rats6. Oxygen free radicals play an important role in cerebral ischemia. Vascular reperfusion, subsequent to transient occlusion of carotid artery results in worsening of the histopathological damage if reperfusion is achieved after some critical period of occlusion (i.e. ischemia)7. The oxygen free radicals initiate lipid peroxidation and inflict damage on

macromolecular components of the cell7,8. In contrast to acute ischemia-reperfusion, a chronic partial/total reduction in cerebral blood flow and brain energy metabolism causes behavioral and cognitive defects9−12. In view of the reported antiinflammatory, antioxidant, cognition-enhancing and antistress properties of P. pinnata6,3,4, it will be of interest to investigate its effect on cerebral ischemia-reperfusion injury and long-term hypoperfusion. Materials and Methods Drug and reagent⎯1, 1, 3, 3-Tetraethoxypropane (TEP, Merck, Germany), thiobarbituric acid (TBA), NADH, nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), (Sigma, USA) were used. All other chemicals and reagents were of the highest analytical grades available locally. Pharmacognostically identified roots of P. pinnata were obtained from Regional Research Institute (Ayurveda), Patna. A voucher specimen has been kept

___________ *Correspondent author Present address: Department of Pharmacology, SGRR Institute of Medical & Health Sciences, Dehradun 248 001, India Telephone: +91 1352522181; Fax: +91 1352721094 E-mail: [email protected].

RAGHAVENDRA et al.: EFFECT OF ETHANOLIC EXTRACT OF PONGAMIA ROOT

869

in the Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University. A sample of P. pinnata root (900 g) was crushed, powdered by grinding, extracted with ethanol and then concentrated under steam bath. The yield was 1.11% (9.99 g of dried extract was obtained at the end). The extract tested positive for steroids, glycoside and flavonoids (done at Indian Herbs Ltd, Saharanpur). Animals received this extract added to a suspension of 1% gum acacia in double distilled water at a dose of 50 mg kg-1 d-1 po. This particular dose was chosen on the basis of earlier reports3.

Animals⎯The present study was conducted on inbred Charles Foster (CF) male albino rats (18 months old) weighing 250-300 g, obtained from the central animal house of the Institute of Medical Sciences, Banaras Hindu University, Varanasi. They were kept in the departmental animal house in colony cages at an ambient temperature of 25°±2°C and 45-55% RH with 10:14 hr light: dark cycles. They had free access to standard rodent pellet diet and drinking water and were fasted for 18-24 hr before surgery (water was allowed ad libitum). The study protocol was approved by the Institutional Ethical Committee and the Principles of laboratory animal care guidelines (NIH Publication No. 86-23, revised 1985) were followed throughout.

Experimental procedure⎯The surgical technique used in the present experiments for induction of cerebral ischemia by bilateral common carotid artery occlusion (BCCAO) was adapted from the earlier published method of Iwasaki et al.13. Rats were anaesthetized by ketamine (100 mg kg-1, ip). Both common carotid arteries were carefully exposed through a midline skin incision in the neck. Acute ischemia-reperfusion injury was produced by blocking bilateral common carotid arteries (BCCA) for 30 min (lifting arteries with loose ligature with the help of thread) and reperfusion for 45 min was allowed by just untying the ligature and releasing the thread. Body temperature was maintained at about 37°C during the period with the help of a heating lamp. This protocol was adopted on the basis of earlier reports8,14. At the end of reperfusion injury, the animals were sacrificed by decapitation and the frontoparietal part of cerebral cortex from both the hemispheres were dissected out and transferred to appropriate homogenizing medium for biochemical estimation.

For long-term hypoperfusion studies, BCCAs were doubly ligated with 3-0 silk sutures and cut in between15. The skin was then sutured and animals were returned to their home cage. On the day 15, 60 min after the last dose of P. pinnata, all animals were subjected to behavioral testing and histopathological studies. For acute studies, the animals were divided into 4 groups of 6 animals each. Group I served as sham-operated control (underwent all surgical procedure except BCCAO). In Group II, P. pinnata (50 mg kg−1d−1, po) was administered to sham-operated animals to determine the effect of drug per se. Group III animals underwent 30 min BCCAO and 45 min reperfusion. In Group IV (treatment) animals, P. pinnata was administered 60 min before subjecting the animals to ischemia-reperfusion and thereafter continued for 5 days in the dose of 50 mg kg-1 d-1, po. For the long-term hypoperfusion studies, animals were again divided into 4 groups of 6 animals each. First group served as sham-operated control. In second group, P. pinnata (50 mg kg-1 d-1, po) was administered during the experimental period in sham-operated animals (per se). Third group animals underwent permanent BCCAO and received vehicle only (hypoperfusion group). In the fourth group, P. pinnata was administered 60 min before BCCAO. P. pinnata was then continued upto 15th post surgical day in sham-operated per se and hypoperfused (treatment group) animals. On day 15 (60 min after the last dose of drug in drug treated groups), animals of all groups were subjected to behavioral assessment paradigm and subsequently they were sacrificed by guillotine and brain samples were collected for histopathological studies.

Biochemical analysis⎯At the end of experiments animals were sacrificed by decapitation and the frontoparietal part of the cerebral cortex from both the hemispheres were separated. After rinsing with ice-cold normal saline, the brain tissue were transferred to the appropriate homogenizing medium and analyzed for the biochemical parameters of the oxidant-antioxidant status i.e. thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD) activity and tissue total sulfhydryl (T-SH) level. All the procedures on the brain samples were performed on ice or ice bath and samples were kept in the freezer compartment (−20°C) of refrigerator. All biochemical parameter were measured in the

INDIAN J EXP BIOL., OCTOBER 2007

870

frontoparietal part of the cerebral cortex of both the hemispheres. Lipid peroxidation⎯Estimation of lipid peroxidation was done by measuring the lipid peroxidation product TBARS (thio barbituric acid reactive substances) following the method of Ohkawa et al.16. TEP was used as an external standard and the level of lipid peroxidation was expressed as nanomoles TBARS mg-1 of protein. Superoxide dismutase (SOD)⎯SOD was estimated by adopting the procedure of Kakkar et al.17. The inhibition by SOD of reduction of nitroblue tetrazolium to blue colored chromogen in presence of phenazine methosulphate and NADH was measured at 560 nm. One unit of enzyme activity was defined as enzyme concentration required to inhibit the absorbance at 560 nm of chromogen production by 50% in one min under assay condition and expressed as specific activity in milliunits mg-1 of protein. Total tissue sulfhydryl groups (T-SH)⎯Total T-SH groups in brain were measured following the method of Sedlack and Lindsay18. The level of T-SH groups was expressed as moles of SH 100-1 g of wet tissue weight. Estimation of brain protein⎯The protein content of brain tissue was estimated using the method of Lowry et al.19. Behavioral testing Open field test⎯Locomotor activity was evaluated in an open field paradigm20. The open field was made of plywood and consisted of a floor (96 × 96 cm) with high walls (61 × 61 cm). Entire apparatus was painted black except for 6 mm thick white lines that divided the floor into 16 squares. The entire room except the open field was kept dark during the experimentation. The open field was lit by a 60 watt bulb focusing on to the field from a height of 100 cm from the floor. Each animal was placed individually in one corner of the apparatus and for the next 5 min, it was observed for ambulations (number of squares crossed), total period of immobility (in sec), number of rearings, groomings and fecal pellets. Morris’ water maze test⎯The maze consisted of a black circular pool21 (diameter 2.14 m, height 80 cm) filled to a depth of 44 cm with water (25°C±1°C). Water was made opaque by adding Indian ink. On day 15 after surgery, spatial learning and memory was tested in water maze. On 14th day the rats received habituation (exposure in water maze for 1 min) in

which there was no platform present. Then, on day 15, a circular platform (9 cm in diameter) was kept hidden 2 cm below water level in the center of one of the quadrants. The platform remained in the same position during training days (reference memory procedure) (escape trial). At the beginning of each session, a random sequence of four starting poles along the perimeter of the pool was generated. All animals followed this sequence for that session. Each rat was placed in the water facing the wall at the start location and was allowed 90 sec to find the hidden platform. The animal was allowed a 20 sec rest on the platform. The latency to reach the platform was recorded. If the rat was unable to locate the hidden platform, it was lifted out and placed on the platform for 20 sec. The procedure was repeated for all the four start locations. Two sessions of 4 trials each were conducted on first day of testing separated by 4 hr and one session of 4 trials was conducted on the next day. After that, the platform was removed and a probe trial (without platform) was conducted 4 hr later. Each rat was placed in the pool at the same randomly selected starting pole and swimming path was observed and time spent in the quadrant of pool which initially contained platform was recorded. On completion of the probe trial, a black platform that extended 1 cm above the surface of water was placed in a quadrant other than that chosen for the submerged platform. Each rat was then given 4 trials of 90 sec to locate it. The latency to reach the platform was recorded (working memory procedure). Histopathological examinations⎯At the end of experimental procedures and behavioral testing, rats were sacrificed and the brains were taken out. The brains were transferred to 10% formalin. Sections (<5 µm thick) of the frontal forebrain were prepared and stained by hematoxylin and eosin for microscopical examination. Statistical analysis⎯Statistical analysis was performed by one-way Analysis of Variance (ANOVA) followed by post hoc Tukey Test for biochemical parameters and behavioral observations. A P-value of <0.05 was considered statistically significant. Results Biochemical Observations⎯Acute BCCAO for 30 min followed by 45 min reperfusion induced a 2-fold increase in lipid peroxidation (TBARS), 2.1-fold SOD


871

activity and a 35% fall in T-SH levels. Pretreatment of rats subjected to cerebral ischemia-reperfusion injury with P. pinnata root extract attenuated the increases in lipid peroxidation (P <0.01) and SOD levels (P <0.01) and prevented the fall in T-SH (P <0.05) (Table 1). Behavioral observations—Open field test: Animals with permanent BCCAO (hypoperfusion group) showed marked alterations in locomotor activities in open field paradigm. Permanent BCCAO was associated with reduced number of ambulations (P <0.01), rearings (P <0.05) and groomings (P <0.01) along with an increase in the period of

immobility (P <0.01) (Table 2). P. pinnata pretreatment prevented these alterations. P. pinnata per se did not have any effect on any of the parameters of the test (Table 2). Morris’ water maze test: Table 3 shows that there was no difference between sham-operated control and P. pinnata per se groups with respect to any of the parameters. All rats were able to locate the hidden platform during the sessions of escape trial. The hypoperfused animals took more time than sham-operated control in finding submerged platform. The increases in the time taken in finding the platform were significant in both the second and third trials

Table 1⎯Effect of P. pinnata (50 mg kg-1 po for 5 days) on biochemical parameters of oxidative stress in rat forebrain following cerebral ischemia -reperfusion injury (30 min BCCAO followed by 45 min reperfusion injury)

[Values are mean ± SE from 6 rats in each group] Groups TBARS (nM mg-1 protein) SOD milliunits mg-1 protein T-SH (×10-5 M mg-1 protein) I Sham-operated 1.93 ± 0.13 340.92 ± 42.19 3.73 ± 0.20 II. Per se 1.95 ± 0.10 369.18 ± 35.29 3.80 ± 0.43 III Ischemia-reperfusion 3.94 ± 0.26** 730.30 ± 65.29** 3.31 ± 0.12** IV P. pinnata extract treated 2.20 ± 0.15** 490.70 ± 61.49** 3.31 ± 0.12*

F (3, 20) 32.99 11.41 6.25 Gr. I and Gr. IV are compared with Gr. III. Gr. II is compared with Gr. I. P values: * <0.05; ** <0.01 (ANOVA followed by Tukey test).

Table 2⎯Effect of P. pinnata (50 mg kg-1 po for 5 days) on open field parameter in long-term hypoperfused rats [Values are mean ± SE from 6 rats in each group]

Groups Ambulations

(number) Immobility (s) Rearing

(number) Grooming (number)

Fecal pellets (number)

I Sham-operated control 62.83 ± 4.76 26.00 ± 2.00 25.16 ± 1.70 7.50 ± 0.42 3.40 ± 1.21 II Per se 64.50 ± 5.11 26.50 ± 1.75 25.83 ± 1.92 7.83 ± 2.50 2.50 ± 0.75 III Hypoperfusion 30.00 ± 5.55** 58.33 ± 1.52** 18.83 ± 1.20* 4.00 ± 0.58** 2.16 ± 0.40 IV P. pinnata extract treated 68.33± 5.18** 29.00 ± 2.60** 29.16 ± 1.81** 5.83 ± 0.75* 2.83 ± 0.65 F (3, 20) 9.03 75.12 6.57 9.16 Not significant Gr. I and Gr. IV are compared with Gr. III. Gr. II is compared with Gr. I. P values: * <0.05; ** <0.01 (ANOVA followed by Tukey test).

Table 3⎯Effect of P. pinnata (50 mg kg-1 po for 5 days) on learning and memory in long-term hypoperfused rats in Morri’s water maze

[Values are mean ± SE from 6 rats in each group]

Escape latency (Sessions) Groups I II III Probe trial New platform trial I Sham-operated control 65.33 ± 5.66 24.33 ± 2.40 26.50 ± 1.76 34.16 ± 4.00 24.00 ± 2.26 II Per se 62.83 ± 5.13 20.66 ± 2.51 28.16 ± 1.47 35.00 ± 3.80 24.83 ± 2.74 III Hypoperfusion 69.66 ± 4.72 42.00 ± 2.07** 41.33 ± 2.33** 26.33 ± 2.30* 35.83 ± 2.31** IV P. pinnata extract treated 64.83 ± 5.42 26.83 ± 2.00** 20.83 ± 0.79** 36.50 ± 4.75* 20.16 ± 0.94** F (3, 20) Not significant 26.08 26.35 3.96 9.54 Gr. I and Gr. IV are compared with Gr. III. Gr. II is compared with Gr. I. P values: * <0.05; ** <0.01 (ANOVA followed by Tukey test).


872

(P < 0.01). Treatment with P. pinnata prevented this delay in escape latencies in second and third sessions (P < 0.01) but not during the first session. The data of probe trial revealed that the hypoperfused rats spent less time in quadrant of former platform than did the sham-operated rats (P < 0.05). This change was significantly reversed by P. pinnata treatment (P < 0.05). The hypoperfused animals took a longer time to locate the new platform than the sham-operated rats (P < 0.01). P. pinnata treated animals took less time in finding the new visible platform than the hypoperfused animals (P < 0.01). Histopathological observations Acute cerebral ischemia-reperfusion injury⎯Fig. 1 shows normal forebrain architecture of sham-operated control animals (the forebrain architecture obtained in the sections of those rat brain samples in which no

changes are expected as these animals did not undergo any ischemia-reperfusion injuries and hence treated as normal control histological pictures against which the alterations in other groups are compared). When the animals were subjected to 30 min BCCAO followed by 45 min reperfusion there was alteration in forebrain histology in the form of inflammatory cell infiltration (Fig. 3). P. pinnata per se did not produce any change in the histological architecture of forebrain (Fig. 2). P. pinnata (50 mg kg-1, po × 5 days) partially reduced this acute injury induced change in architecture (Fig. 4). Long-term hypoperfusion injury⎯Fig. 5 shows the normal histological picture of the brain in sham-operated controls. Long-term hypoperfusion produced marked alterations in the form of increased size and number of glial cells with nuclear enlargement and inflammatory changes resulting from lymphocytic

Figs 1-4⎯Acute cerebral ischemia-reperfusion injury. Histological appearance of the forebrain. 1 – Sham–operated control animals. Note the normal histology of the brain. 2 – P. Pinnata per se group. Note the normal histology of the brain. 3 – Subjected to 30 min BCCAO followed by 45 min reperfusion. Note the reactive changes in the form of mild inflammatory cell infiltration. 4 – Reperfused forebrains treated with P. Pinnata. Note the partial reduction in the injury induced reactive changes (H & E, 100×).


873

infiltration and cellular edema (Fig. 7). P. pinnata per se did not produce any change in the histological structure of the forebrain (Fig. 6). P. pinnata treatment partially reduced these hypoperfusion-induced changes as reflected by less lymphocytic infiltration and reduced glial cell proliferations (Fig. 8).

Discussion The BCCAO for 30 min followed by 45 min reperfusion causes ischemia-reperfusion biochemical injury. Generation of free radicals initiates lipid peroxidation that is reflected as increased level of TBARS. Increased SOD activity, a marker of oxidative stress, also suggests the same. The

biochemical, behavioral and histopathological observations of this study are in agreement with those reported earlier8,14. Similarly, a fall in T-SH levels reflects consumption of tissue thiols. As such, a fall in GSH (a non-protein sulfhydryl) levels during cerebral reperfusion injury is well reported22. Sulfhydryl compounds are among the most important endogenous antioxidants. They have a role in the maintenance of cellular proteins and lipids in their functional states. When these are consumed, the toxic effects of oxidative insult are exacerbated resulting in increased membrane and cell damage23.

The data obtained in the present study showed that the P. pinnata root extract antagonized ischemia-

Figs 5–8⎯Long-term hypoperfusion injury. 5 – Sham-operated control animals. Note the normal histology of the brain. 6 − P. Pinnata per se group. Note the normal histology of the brain. 7 − Subjected to long-term hypoperfusion. Note the reactive changes in the form of gliosis, astrocytosis, cellular edema and lymphocytic infiltration. 8 − Long-term hypoperfusion brains treated with P. Pinnata. Note the partial reduction in hypoperfusion-induced reactive changes (H & E, 100×).


874

reperfusion injury induced raised TBARS levels. Similarly, the extract also reversed the ischemia-reperfusion induced changes in SOD and T-SH activities. These findings support the earlier observation of Annie et al.5 where flowers of P. pinnata were shown to exert a protective role against cisplatin and gentamycin-induced renal injury and this protective role was explained on the basis of the antioxidant action of the plant. The different extracts of P. pinnata are known to contain flavonoids; and flavonoids have been reported to possess antioxidant property5,24. The observed beneficial effect of P. pinnata on acute cerebral ischemia-reperfusion injury-induced changes in biochemical parameters may thus be attributed to its flavonoids contents. Production of inflammatory cytokines and recruitment of circulatory polymorphs during reperfusion7,25 are well documented. These polymorphonuclear leukocytes are a source of superoxide anions26. Earlier reports showed that ethanolic extract of P. pinnata roots has potent anti-inflammatory activity (Comparable to that of phenylbutazone) in carrageenin and PGE1-induced edema models3,4. The protective role of P. pinnata in reperfusion injury may be attributed to its anti-inflammatory activity.

Long-term hypoperfusion studies have been subjected to critical appraisal, through behavioral analysis27,20,21. The long-term hypoperfused rats in the open field paradigm showed significant reductions in ambulations, rearings and groomings as compared to sham-operated animals. This suggests a propensity towards anxiety and listlessness. P. pinnata significantly prevented the long-term hypoperfusion-induced anxiety. Hypoperfused animals also showed deficits of spatial learning and memory as indicated by Morris’ Water Maze data. This is in accordance with earlier reports of ischemia-induced disturbances of spatial learning and memory27. Hypoperfused animals consistently showed longer escape latencies suggesting a defective registration of task (learning). Moreover, probe trial and new platform trial show deficits of reference and working memory in hypoperfused rats. P. pinnata alleviated the changes in long-term hypoperfusion-induced anxiety and listlessness, along with an improvement of learning and memory deficits. It is not out of place to mention that P.pinnata is known to have nootropic activity28 and it modifies the perturbed central cholinergic

markers of cognition in experimentally demented rats6.

Histopathological observations made in the present study revealed that acute and long- term BCCAO did not lead to extensive ischemic neuronal changes (such as neuronal cell death). This is consistent with earlier reports29,14. The absence of neuronal loss/death needs consideration. Glial activation, when it occurs, has consistently been shown to precede overt neuronal death30,31. Therefore, it is likely that, damage may be revealed if BCCAO is extended beyond this time point. Alternatively, the subtle histological damage would have been apparent if more sophisticated techniques of evaluation like immunohistochemistry or electron microscopy were used32. Further work is needed to arrive at a final conclusion in this regard. The histopathological findings as observed in the present study are not consistent with the outcome of behavioral analysis. Indeed, it is known that functional deficits may result from transmission failure rather than from ultimate energy failure and/neuronal loss33,34,35,29. Behavioral decline in the absence of overt histological damage and disturbance of cerebral energy parameters has already been reported29,34,36,14. However, the observed reactive changes, that is gliosis, recruitment of macrophages/lymphocytes and perivascular inflammation support the reported ischemia and/ or hypoperfusion-induced “reactive microgliosis”37,38,14. The present data showed that P. pinnata had partially prevented these reactive changes.

Permanent BCCAO in rats has been used as one of the animal models for neurodegenerative conditions and dementia32−34,39. Reduced blood flow to the extent of 30-45% in the cerebral cortex and 20% in hippocampus has been observed 1 week after permanent BCCAO in rats. In addition, reduced glucose utilization by 20-30% and 15% in cortex and hippocampus, respectively11,12 was also observed. Chronic reduction in cerebral blood flow and brain energy metabolism leads to progressive cognitive deficits9. Reduced brain energy metabolism causes reactive astrocytosis and microglial activation38. Bioflavonoids are known vasodilators40. Therefore, P. pinnata by virtue of its bioflavonoid contents is likely to improve cerebral perfusion and microcirculation after long-term BCCAO. This in turn may attenuate behavioral disturbances and reactive changes.


875

The present study provides additional evidence concerning the anti-oxidative stress and cognition-enhancing property of P. pinnata. Acknowledgement Thanks are due to Dr. Satish Chauhan, Indian Herbs Ltd, Saharanpur, for quantization of active principle of P .pinnata. References 1 Bhavaprakash Nighantu of Guduchyadi Varga, Hindi

Commentary by Chunekar K C & Pandey G S (Chaukhambha, Varanasi), 1969, 350.

2 Thakur R S, Puri H S & Hussain Akhtar, Major medicinal plants of India (CIMAP, Lucknow, India) (1989) 421.

3 Singh R K, Nath G, Goyal R K & Acharya S B, Pharmacological actions of Pongamia pinnata roots in albino rats, Indian J Exp Biol, 35 (1997) 831.

4 Singh R K, Pandey B L, Antiinflammatory activity potential of Pongamia pinnata root extracts in experimentally induced inflammation in rats, J Basic Appl Biomed, 34 (1996) 1204.

5 Annie Shirwaikar, Malini S & Chandrika S, Protective effect of Pongamia pinnata flowers against cisplatin and gentamycin induced nephrotoxicity in rats, Indian J Exp Biol, 41 (2003) 63.

6 Kumar A, Singh R K, Effect of Pongamia pinnata on perturbed central cholinergic markers of cognition in experimentally demented rats, J Basic Appl Biomed, 4 (1996) 43.

7 Gringo J M, Reperfusion injury, Transplant Proc, 29 (1997) 59.

8 Nakashima M, Niwa M, Iwai T & Uematsu T, Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rats, Free Radic Biol Med, 26 (1999) 722.

9 Beal M F, Hyman B T & Koroshetz W, Do defects in mitochondrial metabolism underlie the pathology of neurodegenerative diseases? Trends Neurosci, 16 (1993) 125.

10 Martinez G, Di Giacomo C & Carnazza M L, MAP2 synaptophysin immunostaining in rat brain and behavioral modifications after cerebral postischemic reperfusion, Dev Neurosci, 19 (1997) 457.

11 Tsuchiya M, Sako K, Yura S & Yonemasu Y, Cerebral Blood flow and histopathological changes following permanent bilateral carotid artery ligation in Wistar rats, Exp Brain Res, 89 (1992) 87.

12 Tsuchiya M, Sako K, Yura S & Yonemasu Y, Local cerebral glucose utilization following acute and chronic bilateral carotid artery ligation in Wistar rats: relation to changes in local cerebral blood flow, Exp Brain Res, 95 (1993) 1.

13 Iwasaki Y, Ito S, Suzuki M, Nagahori T, Yamomoto T & Konno H, Forebrain ischemia induced by temporary bilateral common carotid artery occlusion in normotensive rats, J Neurol Sci, 90 (1989) 155.

14 Yanpallewar S U, Hota D, Rai S, Kumar M & Acharya S B, Nimodipine attenuates biochemical, behavioral and histopathological alterations induced by acute transient and long-term bilateral common carotid occlusion in rats, Pharmacol Res, 49 (2004) 143.

15 Pappas B A, De La Torre J C, Davidson C M, Keyes M T & Fortin T, Chronic reduction of cerebral blood flow in the adult rat: late emerging CA 1 cell loss and memory dysfunction, Brain Res, 708 (1996) 50.

16 Ohkawa H, Ohishi N & Yagi K, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal Biochem, 95 (1979) 351.

17 Kakkar P, Das B & Viswanathan P N, A modified spectrophotometric assay of superoxide dismutase, Indian J Biochem Biophy, 21 (1984) 130.

18 Sedlack J & Lindsay R H, Estimation of total, protein bound and non-protein bound sulfhydryl groups in tissue with Ellman’s reagent, Anal Biochem, 25 (1968) 192.

19 Lowry O H, Rosenborough N J, Farr A L & Randal R J, Protein measurement with folin phenol reagent, J Biol Chem, 193 (1951) 265.

20 Lister R G, Ethologically based animal models of anxiety disorders, Pharmacol Ther, 46 (1990) 321.

21 Morris R, Development of a water maze procedure for studying spatial learning in rat, J Neurosci Meth, 11 (1984) 47.

22 Panigrahi M, Sadguna Y & Bangalere R, Alpha-lipoic acid protects against reperfusion injury following cerebral ischemia in rats, Brain Res, 217 (1996) 184.

23 Thomas J A, Poland B & Honzatko R, Protein Sulfhydryls and their role in antioxidant function of protein-S-thiolation, Arch Biochem Biophys, 319 (1995) 1.

24 Bramley P M & Pridham J B, The relative antioxidant activities of plant derived polyphenolic flavonoids, Free Radic Res, 4 (1995) 375.

25 Matsuo Y, Onodera H & Shiga Y, Role of cell adhesion molecules in brain injury after transient middle cerebral artery occlusion in rat, Brain Res, 656 (1994) 344.

26 Lucchesi B R, Werns S W & Fantone J C, The role of neutrophils and free radicals in ischemic myocardial injury, J Mol and Cell Cardiol, 21 (1984) 1241.

27 Jaspers R M A, Block F, Heim C, Sontag K H, Spatial learning is affected by transient occlusion of common carotid arteries (2VO): Comparison of behavioral and histopathological changes after ‘2VO’ and ‘four-vessel occlusion in rats, Neurosci Lett, 117 (1990) 149.

28 Dahanukar S A, Kulkarni R A & Rege N N, Pharmacology of medicinal plants and natural products, Indian J Pharmacol, 32 (2000) 81.

29 Plaschke K, Grant M, Weigand M A, Neuromodulatory effect of propentofylline on rat brain under acute and long-term hypoperfusion, Br J Pharmacol, 133 (2001) 107.

30 Gao H M, Jiang J, Wilson B, Zhang W, Hong J S & Liu B, Microglial activation mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson’s disease, J Neurochem, 81 (2002) 1285.

31 Jorgensen M B, Finsen B R, Jensen M B, Castenellano B, Diemer N H, Zimmer J, Microglial and astroglial reaction to ischemic and Kainic acid–induced lesions of the adult rat hippocampus, Exp Neurol, 120 (1993) 70.

32 Sarti C, Pantoni L, Bartolini L & Inzitari D, Persistent impairment of gait performances and working memory after bilateral common carotid artery occlusion in the adult Wistar rat, Behav Brain Res, 136 (2002) 13.


876

33 Ni J M, Ohta H, Matsumato K & Watanabe H, Progressive cognitive impairment following chronic cerebral hypoperfusion induced by permanent occlusion of bilateral carotid arteries in rats, Brain Res, 653 (1994) 231.

34 Plaschke K, Yun S, Martin E, Hoyer S & Bardenheuer H J, Interrelation between cerebral energy metabolism and behavior in a rat model of permanent brain vessel occlusion, Brain Res, 830 (1999) 320.

35 Sontag K H, Heim C & Block F, Cerebral oligemic hypoxia and forebrain ischemia-common and different long lasting consequences, in Pharmacology of cerebral ischemia edited by J Krieglestein & H Oberpichler-Schwank, (Wissenschaftliche Verlagsgeuschaft, Stuttgart), (1992) 419.

36 Ueda M, Muramatsu H, Kamiya T, Muramatsu A, Mori T, Terashi A & Katayama Y, Pyruvate dehydrogenase activity

& energy metabolic levels following bilateral common carotid artery occlusion in rat brain, Life Sci, 67 (2000) 821.

37 Abraham H & Lazar G, Early microglial reaction following mild forebrain ischemia induced by common carotid artery occlusion, Brain Res, 862 (2000) 63.

38 Bloc F, Peters M & Nolden-Koch M, Expression of IL-6 in the ischemic penumbra, Neuroreport, 11 (2000) 963.

39 Tanaka K, Ogawa N, Asanuma M, Kondo S & Nomura M, Relationship between cholinergic dysfunction and discrimination learning disabilities in Wistar rats following chronic cerebral hypoperfusion, Brain Res, 729 (1996) 55.

40 Duarte J, Perez-Vizcaino F, Jimenez J, Zarzuclo A & Tamagro J, Vasodilator effect of quercetin in isolated rat vascular smooth muscle, European J Pharmacol, 239 (1993) 1.

effect of ethanolic extract of root of pongamia pinnata (l...

Documents