|Year : 2011 | Volume
| Issue : 4 | Page : 519-524
Effect of amiloride: An Na + / H + exchange inhibitor in the middle cerebral artery occlusion model of focal cerebral ischemia in rats
Mohammad Akhtar, KK Pillai, Abul Kalam Najmi, Divya Vohora
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, New Delhi, India
|Date of Submission||02-Feb-2011|
|Date of Decision||26-Mar-2011|
|Date of Acceptance||06-May-2011|
|Date of Web Publication||23-Nov-2011|
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, New Delhi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Purpose : The effect of pretreatment with amiloride (AML), an Na + / H + exchange inhibitor was studied in the middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats. Materials and Methods : Male wistar rats were subjected to 2 hr of MCAO followed by 22-hr reperfusion. Grip strength, locomotor activity, and spontaneous alternation performance were assessed after 24 hr. Immediately after behavioral activities, animals were sacrificed and the oxidative stress markers were estimated in brains. Results : An elevation of thiobarbituric acid reactive substances (TBARS), reduction in glutathione, and antioxidant enzymes activities, namely glutathione-S-transferase, glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD) were observed following MCA occluded rats. Pretreatment with AML (0.91 and 1.82 mg/kg p.o) significantly reversed the MCAO-induced elevation in TBARS but could not reverse the other parameters. Paradoxically, AML further reduced the levels of GPx, GR, and SOD, but no significant changes were observed in the catalase activity, grip strength, and spontaneous alternation behavior of rats. Locomotor activity was reduced slightly but reversed on pretreatment with AML. Conclusions : Although pretreatment with single dose of AML showed reduction in oxidative stress markers, further multiple doses of AML as pre- and post-treatments are required to establish its potential to be used in cerebral ischemia.
Keywords: Amiloride Na + / H + exchanger, MCA occlusion, oxidative stress
|How to cite this article:|
Akhtar M, Pillai K K, Najmi AK, Vohora D. Effect of amiloride: An Na + / H + exchange inhibitor in the middle cerebral artery occlusion model of focal cerebral ischemia in rats. J Pharm Bioall Sci 2011;3:519-24
|How to cite this URL:|
Akhtar M, Pillai K K, Najmi AK, Vohora D. Effect of amiloride: An Na + / H + exchange inhibitor in the middle cerebral artery occlusion model of focal cerebral ischemia in rats. J Pharm Bioall Sci [serial online] 2011 [cited 2020 Jul 13];3:519-24. Available from: http://www.jpbsonline.org/text.asp?2011/3/4/519/90105
Among the cardiovascular disorders, stroke is a major cause of mortality and long-term disability. Currently there is no effective therapy established yet that can restore the diminished blood supply to the dying neurons or improve the cognitive deficits except thrombolytics.  Ischemia/reperfusion injury is a complex and incompletely understood phenomenon. Normally ischemic tissues are deprived of energy that is important for the maintenance of ionic balance. Reperfusion initiates reoxygenation-induced inflammatory response that further exacerbates injury.  Neurons are more vulnerable to hypoxia/ischemia, as a consequence release of excitatory amino acids, neurotransmitters, and glutamate, activating the receptor-dependent Ca 2+ channel and hence Ca 2+ overloads.  All these processes are responsible for altering the intracellular pH, resulting in cellular acidosis, which in turn causes brain injury.  In the brain, Na + / H + - exchanger (NHE), a ubiquitous protein, participates in variety of processes including restoration of intracellular pH regulation, cell volume, cell proliferation, migration, and cell death via apoptosis or necrosis.  Furthermore, both cell volume and intracellular pH are essential for normal cell functioning; they are regulated by NHE isoform (NHE 1) and Na + K + -2Cl - co-transporter.  NHE1 is widely expressed in the Central nervous system (CNS) and is the most abundant NHE isoform in the cerebral cortex.  NHE1 plays a key role in pH regulation in both neurons and glial cells and may be crucial in both physiological and pathophysiolgical conditions.  Cerebral ischemia is associated with the stimulation of NHE1 activity resulting in neuronal damage.  Luo et al,  reported that NHE1 activity disrupted Na + and Ca 2+ homeostasis and contributed to ischemia neuronal damage. There are several experimental and clinical studies, which demonstrated that NHE plays an important role in the pathogenesis of ischemic brain injury ,,, and their inhibitors may provide cardioprotective and neuroprotective effects; however, the precise mechanisms are ambivalent and elusive. Therefore, this study was designed with the view that pretreatment with amiloride (AML); an NHE1 inhibitor can provide protection of cerebral neurons from ischemia/reperfusion injury induced by middle cerebral artery occlusion (MCAO) in rats, and also improve cognitive functions. Several components of reactive species have been found to be generated after ischemia reperfusion injury; therefore, this study also evaluated the role of AML on oxidative stress markers.
| Materials and Methods|| |
Swiss strain albino male wistar rats (350-400 g) supplied by the Central Animal House Facility of Jamia Hamdard, New Delhi, were used. All animals were housed in cages kept at a temperature of 23-30°C with a natural light - dark cycle. They had free access to standard pellet diet (Amrut Laboratory rat and mice feed, NavMaharastra chakan oil mills Ltd, Pune) and tap water. The study was approved by Animal Ethics Committee CPCSEA (Project no 134). Ethical norms were strictly followed during all experimental procedures.
Drugs and dosing schedule
AML was procured from Micro Nova Pharmaceutical, India, dissolved in double distilled water at a dose of 0.91 and 1.82 mg/kg p.o. Normal control group and sham operated animals received double distilled water as solvent of drug. The dose of AML was selected based on the human dose used therapeutically and converted into animal dose and the same was administered once 2 hr before the MCAO. Animals were divided into five groups. Each group consisting of six rats each receiving different treatments for once dose administration. GP I vehicle control, GP II sham operated, GP III MCAO only, GP IV AML 0.91 mg/kg p.o. + MCAO, GP V- AML 1.82 mg/kg p.o. + MCAO. Ischemia was performed for 2 hr and reperfusion for 22 hr. The rats were studied for behavioral parameters and then sacrificed for the estimation of oxidative stress markers in the rat's brain.
Induction of cerebral ischemia
Rats were anaesthetized with choral hydrate (dissolved in distilled water) at a dose of 400 mg/kg intraperitoneally (i.p.). Core (rectal) temperature was maintained around 37°C throughout the surgical procedure using heating lamp and the thermo-controlled base of the operating table. A midline incision was made and the right common carotid artery, external carotid artery, and internal carotid artery were exposed. A 4.0 monofilament nylon thread (40-3033pk10, Doccol Corporation, Pennsylvania, Ave Redlands CA, USA), the tip of which was rounded, was inserted into the external carotid artery and advanced into the internal carotid artery until a slight resistance was felt. Such resistance indicated that the filament had passed beyond the proximal segment of the anterior cerebral artery. At this point, the intraluminal filament blocked the origin of the MCA and occluded all sources of blood flow from the internal carotid artery, anterior cerebral artery and posterior cerebral artery. The nylon filament was allowed to remain in the place for 2 hr after which it was gently withdrawn until tip reached external carotid artery to allow the reperfusion of the ischemic region.  In sham-operated rats, the external carotid artery was surgically prepared for the insertion of the filament, but the filament was not inserted. Ischemia was performed for 2 hr and reperfusion for 22 hr. After 24 hr, the rats were then studied for motor performance tests and the spontaneous alternation behavior. Immediately after behavioral tests the animals were then sacrificed their brain removed, homogenate was prepared and biochemical estimations were carried out.
Motor performance tests
Locomotor activity (Closed field activity monitoring)
pontaneous locomotor activity of rats was measured as described by Lannnert et al,  using digital photoactometer (Techno, India). Each rat was observed for a period of 10 minutes in a square closed arena equipped with infrared light (IR) sensitive photocells. When the animal crosses the beam of IR light, a count is recorded and displayed on the monitor. During activity testing, only one animal was tested at a time.
Grip strength test
Grip strength meter was used as described by Ali et al,  for recording the grip strength of the animal. Briefly, the rats were allowed to hold the grid, which was then pulled back horizontally until the rat released its grip. The force achieve by animal was than displayed on the screen and was be recorded as Kg unit.
Spontaneous alternation behavior
Spontaneous alternation behavior (SAB) was performed in a plus maze to assess effects of drugs on short term memory with respect to spatial orientation and perception as described by Ragozzino et al,  was used. The rats were placed in a four-arm cross maze. The maze (85 cm height) was constructed of wood painted gray and contained a central platform (25 cm diameter), from which radiated four symmetrical arms (55 cm long and 10 cm wide), with 12 cm wall. After being placed in the central platform, rats were allowed to traverse the maze freely for 12 minutes. The number and sequence of entries were recorded. An alternation was defined as entry into four different arms on an overlapping quintuple set. Five consecutive arm choices within the total set of arm choices constitute a quintuple set. A quintuple consisting of arm choices A, B, A, C, D was considered as an alternation while the set with A, B, A, C, B did not. Using this procedure percentage alternation is equal to the ratio of (actual alternation to possible alternation) X 100. Possible alternation sequences are equal to the number of arm entries minus 4.
Estimation of oxidative stress markers
Following the behavioral testing, the animals were decapitated and the brains were quickly removed, sliced, weighed, and homogenized in ice cold KCl phosphate buffer (0.1 M pH 7.4) 10 times (w/v) and centrifuged at 2000 rpm for 5 min at 4°C. The supernatant containing crude membrane were used for the estimation of TBARS and GSH. The remaining supernatant was again centrifuged at 10 000 rpm at 4°C for 20 min. The postmitochondrial supernatant was used for the study of antioxidant enzyme activities and protein estimation. Catalase and Superoxide dismutase activities were determined immediately after sample preparation. Protein concentrations were determined according to Lowry et al,  using purified bovine serum albumin as standard.
Measurement of lipid peroxidation
Thiobarbituric acid reactive substance (TBARS), a measure of lipid peroxidation was measured as described by Ohkawa et al.  Briefly, 0.1 ml homogenate, 1 ml of trichloroacetic acid (10%) and 1 ml of thiobarbituric acid (0.67%) were added to all test tubes, covered with aluminum foil and placed in boiling water bath for 20 min. Test tubes were then shifted to crushed ice bath and then centrifuged at 6000 rpm for 10 min. The absorbance of the supernatant was measured at 540 nm. TBARS values were expressed as n moles MDA/mg protein.
Measurement of reduced glutathione
Glutathione was measured according to the method of Ellman.  The equal quantity of homogenate (w/v) and 10% trichloroacetic acid were mixed and centrifuged to separate the proteins. To 0.01 ml of this supernatant, 2 ml of phosphate buffer (pH 7.4), 0.5 ml 5, 5'-dithiobisnitro benzoic acid (DTNB) and 0.4 ml of double distilled water was added. The mixture was vortexed and the absorbance was read at 412 nm within 15 min. GSH values were expressed as ΅ moles GSH mg protein.
Measurement of catalase
Catalase activity was measured by the method of Claiborn.  A total of 0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7). The reaction was started by the addition of 1 ml freshly prepared 30 mM H 2 O 2 . The rate of decomposition of H 2 O 2 was measured spectrophotometrically at 240 nm. Catalase values were expressed as n moles H 2 O 2 consumed/min/mg protein.
Measurement of Glutathione S- transferase (GST)
GST activity was measured by the method of Habiq et al.  The reaction mixture consisted of 1.425 ml of phosphate buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0.025 ml CDNB (1mM), and 0.3 ml PMS (10% w/v) in a total volume of 2 ml. The change in absorbance was recorded at 340 nm. GST values were expressed as n moles CDNB conjugate formed/min/mg protein.
Measurement of glutathione peroxidase (GPx)
Glutathione peroxidase activity was measured according to the procedure described by Mohandas et al.  The reaction mixture consisted of 1.44 ml phosphate buffer (0.05 M, pH 7), 0.1 ml of EDTA (1mM), 0.1 ml sodium azide (1mM), 0.05 ml glutathione reductase (1 enzyme unit/ml), 0.1 ml glutathione (1mM), 0.1 ml of NADPH (0.2 mM), 0.01 ml of H 2 O 2 (0.25 mM), and 0.1 ml of PMS (10% w/v) in a final volume of 2 ml. The disappearance of NADPH at 340 nm was recorded at room temperature. GPX values were expressed as n moles NADPH oxidized/min/mg protein.
Measurement of glutathione reductase (GR)
GR activity was assayed by the method of Calberg and Mannervik,  as modified by Mohandas et al.  The assay system consisted of 1.65 ml of phosphate buffer (0.1 M, pH 7.6), 0.1ml NADPH (0.1mM), 0.1ml EDTA (0.5 mM), 0.05 ml oxidized glutathione (1 mM), and 0.1 ml PMS (10% w/v) in a total volume of 2 ml. The enzyme activity was recorded by measuring the disappearance of NADPH at 340 nm. GR values were expressed as n moles NADPH oxidized/min/mg protein.
Measurement of superoxide dismutase (SOD)
SOD activity was measured by the method of Beauchamp et al.  The reaction mixture of total 1 ml consisted of 0.6 ml of phosphate buffer (0.5 M, pH 7.4), 0.1 ml PMS (10 % w/v), 0.1ml xanthine (1mM), 0.1ml NBT (57 mM) was incubated for 15 min at room temperature and reaction was initiated by the addition of xanthine oxidase (50 mU). The rate of reaction was measured by recording change in the absorbance at 550 nm. SOD values were expressed as n moles formazan formed/min/mg protein.
The results of all the experiments were expressed as Mean + SEM (standard error of mean) and analyzed by ANOVA followed by Dunnett's t-test. P values less than 0.05 were considered significant. It is carried out with graph pad in Stat 3 software.
| Results|| |
Effect of amiloride on the oxidative stress markers in middle cerebral artery occluded (MCAO) rats
TBARS levels were significantly elevated in MCA occluded rats (P<0.01). AML pretreatment (0.91 and 1.82 mg/kg p.o.) followed by MCA occlusion significantly reduced the elevated TBARS levels [F (4, 29) = 13.596, P<0.0001]. MCA occlusion resulted in the significant reduction of glutathione levels as compared with sham group (P<0.01). AML (both doses) pretreatment reversed such change and showed significant elevation of GSH levels [F (4, 29) = 23.884, P<0.0001]. No significant change in catalase activity was observed in response to either MCA occlusion or AML treatment groups [F (4, 29) = 3.057, P<0.352]. MCA occluded and AML-treated groups showed significant reduction of GST levels when compared with control and sham groups [F (4, 29) = 21.730 P<0.0001]. GPx activity was reduced in MCA occluded groups when compared with control (P<0.01). AML (both doses) also showed significant reduction when compared with sham group [F (4, 29) = 7.121, P<0.0006]. The GR activity was reduced, though statistically insignificant (P>0.05) and similar pattern of reduction was also observed at both dose levels of AML [F (4, 29) = 5.789, P<0.0019]. No significant changes were observed in SOD activity in either MCA occluded or AML treated groups [F (4, 29) = 2.810, P=0.471] [Table 1].
|Table 1: Effect of amiloride on the oxidative stress markers in middle cerebral artery occluded rats|
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Effect of amiloride on behavioral parameters in MCAO rats
The locomotor count was reduced in MCA occluded rats though not statistically significant (P>0.05). Pretreatment with AML (0.91 and 1.82 mg/kg p.o.) showed that the locomotor count was returning toward normal. No significant change in grip strength and percentage alternations was observed in control, sham and AML treated groups (P>0.05). However, MCA occluded rats showed increase in grip strength but statistically insignificant. A significant reduction was seen in possible alternations in MCA occluded rats as well as AML (both doses) treated groups when compared with control groups (P<0.05). AML (1.82 mg/kg p.o.) also showed reduction when compared with sham group [F (4, 29) = 8.962, P=0.0001] [Table 2].
|Table 2: Effect of amiloride on behavioral parameters in middle cerebral artery occluded rats|
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| Discussion|| |
The Na + /H + exchanger (NHE) is a group of membrane transport proteins that catalyzes the secondary active electoneutral exchange of one Na + for one H + . To date nine, NHE isoforms (NHE1-9) have been cloned in mammalian tissues. The first identified NHE isoform, NHE1 is ubiquitously expressed on the plasma membrane of virtually all mammalian cell types. NHE1 is a major membrane transport mechanism in regulation of pH i and cell volume.  Neurons are susceptible to injury from acidosis due to their high metabolic rate. During cerebral ischemia there is an alteration of intracellular pH, resulting in acidosis. Emerging evidence points towards the role of NHE1 in intracellular pH regulation under ischemia reperfusion conditions.  AML, a NHE inhibitor was used in the present study to assess its role in cerebral ischemia induced by MCA occlusion for 2 hr followed 22-hr reperfusion, after which the oxidative stress markers were estimated in the whole brain of rats. TBARS levels after 24 hr of MCA occlusion were found to be elevated as compared to sham treatment exhibiting that lipid peroxidation and oxidative stress were involved in the neuronal injury. Besides elevated TBARS levels, a significant reduction in the GSH levels was also observed following MCAO, thus confirming the increase in free radical production in the postischemic period particularly after reperfusion. ,, We also observed a decline in the levels of antioxidant enzymes including GST, GPx, GR, and SOD, though the last two being statistically insignificant.
Pretreatment with AML (at both 0.91 and 1.82 mg/kg p.o) reversed the MCAO induced increase in TBARS. Though a tendency towards an increase GSH was also observed following AML, it was found to be statistically insignificant. The possible mechanism of neuroprotective effect of AML could be due to the suppression of excitotoxicity through inhibition of both Ca 2+ influx and acidification in the neurons. Phillis et al,  reported that 5-(N-ethyl-N-isopropyl) AML protected cerebral neurons from ischemia reperfusion injury and implicated that Na + /H + exchange was a causative factor in such injury. Similar neuroprotection in MCAO models were observed using other NHE1 inhibitors including SM-20220;  FR- 183998  and sabiporide.  Giving SM-20220 intravenously 1 hr after 2 hr MCAO significantly reduced the extent of cerebral edema, Na + content and infarct volume following ischemia and reperfusion. Importantly, SM 20220 decreased infarct size in permanent MCAO models even with delayed treatment.  Similarly, another NHE inhibitor sabiporide also decreased infarct size and edema volume at 24 hr reperfusion not only in preischemia treated but also in postischemia treated rats.  A large number of experimental and clinical studies supported the protective effect of NHE inhibitors in ischemic injury. ,,, Recently Kendall et al,  found that blockade of NHE1 with N-methyl-isobutyl amiloride ameliorated brain injury under hypoxia/ischemia in neonatal mice. The effectiveness of NHE1 inhibitors from these studies suggests that they may be useful for stroke treatments of in-post-ischemia administration. Our study provides one such possible mechanism for the protective effect of AML against cerebral ischemia. Contrary to the effects on TBARS and GSH, AML (at both the doses), in our study, could not reverse the reduced levels of antioxidant enzymes. Paradoxically, it further reduced some of these enzymes activities including GPx, GR, and SOD. While the reason for such an effect observed by AML is not clear, one possible explanation for insignificant reduction could be due to a delayed decline of such antioxidant enzymes following MCA occlusion in the groups treated with AML. Lerovet et al,  demonstrated a 48 h decline in the activity of some of the antioxidant enzymes following MCAO. This could be responsible for insignificant reduction in the levels of catalase, GR and SOD following MCAO. Further, the data on SOD has also not been consistent in most of the studies.  Further follow-up studies are required to investigate the exact role and the possible mechanism for the effect of AML in cerebral ischemia. There was no significant change in the grip strength, and percentage alternation of animals following MCAO and AML pretreatment. Locomotor activity was reduced slightly after MCAO but it was reversed on pretreatment with AML.
| Conclusion|| |
To conclude, NHE1 plays an important role in the maintenance of intracellular pH under normal conditions, excessive activation of NHE1 activity appears to be involved in cellular damage under ischemic conditions. Blockade of NHE activity by using selective NHE1 inhibitors could be a novel approach in cerebral ischemia. Although, our results are preliminary using single dose of AML, but we observed reduction in oxidative stress markers by virtue of antioxidant and anti-inflammatory actions of AML. Further studies with multiple doses of AML as pre- and post-treatments are required to establish its possible neuroprotective mechanism and usefulness both as pre and postischemia administration in cerebral ischemia.
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