Effects Of Amiloride On Global Cerebral Ischemia-reperfusion Injury In Rats

Objective: To investigate the effects of amiloride on global cerebral ischemia-reperfusion (I/R) injury in rats.Methods: Forty-eight healthy adult male Sprague-Dawley rats weighing 350-300g were randomly divided into 4 groups (n=12 each): sham operating group (S); global cerebral I/R injury group (I/R); normal saline group (NS) and amiloride group (A). Global cerebral I/R injury was produced by 3-vessel occlusion. The animals were anasthetized with intraperitoneal 10% chloral hydrate 0.3 g·kg-1 and placed in the supine position. The bilateral common carotid arteries (CCAs) were isolated. A craniotomy was then made in the bone at the base of the skull. The dura was incised to expose the basilar artery, threading at the second no-branch region. Then animals were fixed in the stereotaxis instrument for microdialysis. The probe was pricked into brain 3.5 mm at the intersection point behind anterior fontanel 2.0 mm and beside sagittal suture 3.0 mm. The microdialysic fluid had been consecutively collected for 1 h, each tube 10 min. In group S bilateral CCAs and basilar artery were not occluded. 3 arteries had been occluded for 10 min followed by 8 h reperfusion after the first tube in group I/R, NS and A. And in group NS and A, amiloride 0.6 mg/kg or equal volume of NS solution were given at the beginning of the reperfusion through femoral vein. Rats were tested the open fild test (OFT) and the inclined plane test (IPT) and then were killed at the end of 8 h reperfusion. The right brain of 6 rats of each group were removed for light microscopic examation, the left brain for expression of acid-sensing ion channel 2b protein in brain by immunohistochemistry. The brain of the others were removed for electron microscopic examation and determination of brain water content.Result:1 The grade of neuroethologyOFT: The count of grids was significantly littler in group I/R, NS, and A as compared with group S and it was significantly more in group A than in group NS and I/R (P<0.05). There was no significant difference between group NS and I/R (P>0.05). IPT: Compared with group S, the time of turning was longer in group I/R, NS and A and it was shorter in group A as compared with group Na and I/R (P<0.05). While it was not significantly different between group NS and I/R (P>0.05).2 The brain water contentThe the brain water content was increased in group I/R, NS and A as compared with group S but amplitude was significantly smaller in group A than in group I/R and NS (p<0.05). It is not significantly different between group I/R and group NS (P>0.05).3 Pathology of light microscopeGroup S: The morphosis of nerve cells was nearly whole The cytoplasma was plentiful and had been thin dyed. The nucleolus was clear.Group I/R: Most of the nerve cells were wizened and the dyeing aggravated. The nucleolus was unclear. The boundary between the cytoplasma and the nucleolus was not pure, while the nucleolus was unclear.Group NS: Parts of the nerver cells were crinkly. The boundary between the cytoplasma and the nucleolus was not pure, while the nucleolus was unclear.Group A: A part of the never cells were wrinkly, but the structur was approximately normal. Karyon was elliptic and nucleolus was clear.4 Pathology of transmission electron microscopeGroup S: The morphosis of nerve cells was almost intact. A little of critae and membrane of mitochondrium were amalgamation or lost. The rough endoplasmic reticulms had little the phenomenon of degranulation. The Golgi's complex was normal and had some secondary lysosomes and free ribosome.Group I/R: The cytoplasm was extremely dropsical and the count of organelles decreased. The count of organelle was significantly decreasing. The most of critae and membrane of mitochondrium were fused or lost. And the phenomenon of degranulation was very serious in rough endoplasmic reticulms. The Golgi's complex was not quite whole and there were some free ribosomes.Group NS : The cytoplasm was extremely dropsical and the count of organelles decreased. The count of organelle was significantly decreasing. The most of critae and membrane of mitochondrium were fused or lost. And the phenomenon of degranulation was very serious in rough endoplasmic reticulms. The Golgi's complex was not quite whole and there were some free ribosomes but the secondary lysosomes were reduced.Group A: The morphosis of nerve cells was normal on the whole. The cytoplasm was little dropsical. A part of critae and membrane of mitochondrium were fused or lost. And the phenomenon of degranulation was very light in rough endoplasmic reticulms. The Golgi's complex was almost whole. There were some secondary lysosomes and free ribosomes.5 The concentration of lactate of microdialysic fluidThere were significant differences among four groups before and during occlusion (P>0.05).The concentration of Lac in group A, I/R and NS was significantly higher as compared with group S after reperfusion and it was significantly lower in group A than in group I/R and NS (P<0.05), but there were significant differences among group NS and I/R (P>0.05).6 The expression of ASIC2b protein in the brain tissue The cells with expressoin of ASIC2b protein are brown. The grade of HIS was significantly low in group I/R, A, and NS as compared with group S and, it was higher in group A than in group I/R and NS (P<0.05). There was no significant difference between group I/R and NS (P>0.05).Conclusion: Amiloride has protective effects against global cerebral I/R injury in rats.