acute cerebral ischemic reperfusion injury ( ciri ) , a hotspot for clinical research , is a pathophysiologic phenomenon commonly encountered in the field of emergency medicine , especially during the perioperative periods . the brain can store so little energy reserves that it is highly sensitive to ischemia and hypoxia . studies have shown that interrupting the cerebral blood flow for 10  s can lead to loss of consciousness . if cerebral blood flow is blocked for more than 5  min , permanent brain damage is inevitable . phosphocreatine ( pcr ) has been used as cardioplegic and cardioprotective agents during cardiopulmonary bypass and also ischemic events . in recent years , some reports showed that pcr improved the outcome after stroke and neonatal hypoxic ischemic encephalopathy . there have been no studies done to investigate the effect of pcr during acute ciri . so we designed this research using acute rat ciri model to observe the effect of pcr and investigate its possible mechanism . all experimental procedures were done in accordance with the guide for the care and use of laboratory animals . all surgical procedures have been approved by the   committee for experimental animals of centre for disease control and prevention of hubei province . 36 male wistar rats weighing 200220  g were acquired from the experimental animal center of hubei province , wuhan , china . 36 wistar rats were randomly divided into 3 groups : sham - operated group ( sham group , n = 12 ) , ischemia - reperfusion group ( i / r group , n = 12 ) ,    pcr preconditioning group ( pcr group , n = 12 ) . ciri rat models were produced by electrocauterizing bilateral vertebral arteries and occlusion of bilateral common carotid arteries using atraumatic clasps . the clasps were released 10  min later and followed by a 48-hour reperfusion . in sham group , pcr 150  mgkg was administered intravenously 60  min before the bilateral common carotid arteries were occluded ; normal saline was administered intravenously in i / r group simultaneously . core body temperatures were monitored with a rectal probe and maintained between 36.5c~37.5c during the whole procedure . 3 rats from i / r group and 1 from pcr group were excluded within 24  h while the remaining ones were regarded as successful models . after routine he staining , pathological changes of the brain tissue were observed under a light microscopy ( 400 ) . the level of cell apoptosis was determined with a roche in situ cell - apoptosis - assay kit with nuclei stained in brown particles ( shanghai runwell technology co. , china ) . 10 high - power fields ( 400 ) were randomly selected , and the number of apoptotic cells was counted for each field . cam activity ( mmoll ) was measured using rat cam elisa kit ( r & d company , us ) ; mda content ( nmoll ) was detected with mda kit ; protein content ( mgl ) in corresponding tissue was detected with coomassie brilliant blue protein assay kit ( both from nanjing jiancheng bio co. , china ) . mda content in the tissue was calculated using the following formula : 
 ( 1)mda  ( nmolmgprot1)=mda  content  ( nmoll1)protein  content  ( mgl1 )  in  corresponding  tissue5 . results were expressed as mean  sd . data were statistically evaluated by one - way anova ( snk ) tests and dunnett 's tests , with the level of significance chosen as p < .05 . in sham group , the cortical neurons are arranged in neat rows with abundant cytoplasm , and the nuclei are round and basophilic . in i / r group , the cytoplasm is light red with uneven distribution and vacuoles , nuclei are condensed . in pcr group , however , the cell structure is normal . most of the neurons have complete membrane integrity and the nuclei are clear ( figure 1 ) . a few tunel - positive cells were observed in the cortex of rats in sham group whereas a large number of tunel - positive cells was observed in the cortex of rats subject to i / r injury ; compared with i / r group , the number of tunel positive cells was significantly reduced in the cortex of pcr group ( figures 2 and 3 ) . the cam activity of i / r group and pcr group was significantly increased at 48 hours after reperfusion compared with sham group whereas cam activity was significantly decreased in the cortex of pcr group compared with i / r group ( figure 4 ) . mda in i / r group and pcr group was significantly increased at 48 hours after reperfusion compared with sham group , but compared with i / r group , mda content was significantly decreased in the cortex of pcr group ( figure 5 ) . rapid exhaustion of energy is one of the important etiological factors of ischemia - reperfusion injury [ 5 , 6 ] . we hypothesized that supplication of an exogenous energy substrate before ischemic reperfusion might be an important logical therapeutic step . pcr is a very important energy substrate , it can go through the blood - brain barrier , even through the cell membrane , to supply energy to cells directly . , we have demonstrated that pcr preconditioning attenuated cell apoptosis and the morphological damage during cerebral ischemia - reperfusion in rats . in ischemia - reperfusion injury brain , the cytoplasm is light red with uneven distribution and vacuoles , and nuclei are condensed under light microscopy after reperfusion for 48  h. compared with i / r group , the number of apoptotic cells was significantly decreased and cerebral ischemia - reperfusion injury was alleviated in pcr group . imbalance of neuronal calcium homeostasis and increase in oxygen free radical are important aggravating factors of cerebral ischemic reperfusion injury . energy deficiency causes intracellular calcium overload and an increase in oxygen free radical . to assess the intracellular ca overload and oxygen free radical level , cam activity and cerebral mda content were determined . it combines with ca reversibly , and regulates transmembrane calcium transportation , absorption , and secretion . an increase in activity of cam means an elevation of intracellular ionized ca concentration , hence causing calcium imbalance in the cells . mda is a stable lipid peroxide end product produced during the oxidation of membrane lipid unsaturated fatty acid by oxygen free radical . the central nervous system is rich in unsaturated fatty acids that interact with oxygen free radical during reperfusion after an ischemic event , which generates a large amount of mda . therefore , mda level indirectly reflects the level of oxygen free radical and the degree of lipid peroxidation in the brain tissue . we found that the cam activity was elevated in i / r group compared with sham group , indicating the increase of ca influx , ca overload , and ca balance disturbance . the high mda content in i / r group suggested that oxidative stress occurred and a large amount of lipid peroxide was produced . thus , there were lower incidences of ca reflux , ca overload , and complex pathological changes induced by ca overload . pcr group also had lower mda level , indicating that pcr induced a significant suppression in the generation of free radical and provided an antioxidative protection to the cell and organelle membranes , hence attenuating cellular necrosis and apoptosis .   during ischemia - reperfusion we have found that pcr preconditioning decreases cam activity , thus preventing calcium overload , reducing the production of oxygen free radical indirectly . as an atp precursor , pcr releases high - energy phosphate bond to synthesize atp , supplying neurons with energy , reducing the production of lactic acid , and maintaining the function of na - ca exchange . it is suggested that pcr preconditioning ensures sufficient atp supply , keeps ca pump working , and sustains ca balance . it can also inhibit the formation of hypoxanthine , thereby reducing the production of oxygen free radicals . the drawback of clinical application of mechanical preconditioning is the difficulty in predicting the onset of any ischemic event . even if it 's predictable , the application of a transient mechanical preconditioning is impractical before cerebral ischemia . therefore , the clinical application of mechanical preconditioning was limited . one of the important etiological factors of ischemia - reperfusion injury is due to the rapid exhaustion of energy [ 5 , 6 ] . therefore , a supply of an exogenous energy substance during ischemic reperfusion may be an important logical therapeutic step . it acts as an energy reserve , and it is mainly synthesized by the kidneys . the finding of the current study provides a promising method for the treatment of cerebral ischemia - reperfusion injury . future study will be needed to further evaluate the effect of pcr on brain infarct size , neurological score and so forth . the assessment of functional and histological endpoints as well as multiple neurological outcomes will allow better understanding of the pcr 's role in brain ischemia . in summary , we demonstrated the protective effect of pcr during cerebral ischemic reperfusion injury . based on the pharmacokinetic characteristics of pcr , we speculate that pcr should be administered preoperatively to hemodynamic unstable patients , such as aortic aneurysm surgical patients , who may develop cerebral ischemia during surgery when the major arteries are clamped . other patients with severe head injury , hemorrhagic shock , cerebral vasospasm , and respiratory and cardiac arrest
phosphocreatine ( pcr ) is an endogenous compound containing high - energy phosphate bonds . 
 it has been confirmed that pcr is effective in preventing and treating cardiac and renal ischemia - reperfusion injury . in this study , 
 rat cerebral ischemia - reperfusion injury models were constructed . 
 apoptotic cells in the cortex region were measured by tunel method . 
 malondialdehyde ( mda ) content was detected by chromatometry , and calmodulin ( cam ) activity was detected by elisa . 
 compared with sham - operated group ( sham group ) , tunel - positive cells , mda , and level of cam activity increased in ischemia - reperfusion group ( i / r group ) and pcr preconditioning group ( pcr group ) ; compared with i / r group , tunel - positive cells , mda content , and level of cam activity decreased in pcr group . 
 this study indicated that pcr can decrease the morphological damage and the neuron apoptosis of the ischemia - reperfusion injury brain through attenuating abnormalities of calcium balance and production of oxygen free radicals .