Phosphocreatine (PCr) is a natural compound, which can donate high-energy phosphate group to ADP to synthesize ATP, even in the absence of oxygen and glucose. At present, it is widely used in cardiac and renal ischemia-reperfusion (IR) disease. In this study, to examine the protective efficacy of PCr against cerebral IR, disodium creatine phosphate was injected intravenously into rats before focal cerebral IR. Intracranial pressure (ICP), neurological score, cerebral infarction volume, and apoptotic neurons were observed. Expression of caspase-3 and aquaporin-4 (AQP4) was analyzed. Compared with IR group, rats pretreated with PCr had better neurologic score, less infarction volume, fewer ultrastructural histopathologic changes, reduced apoptosis, and lower aquaporin-4 level. In conclusion, PCr is neuroprotective after transient focal cerebral IR injury. Such a protection might be associated with apoptosis regulating proteins.
Transient cerebral ischemia-reperfusion (IR) injury is a major complication in stroke, resuscitation, and perioperative period, in which 50–70% survivals suffer from severe disabilities [
Some studies showed that PCr could improve the outcome after neonatal hypoxic ischemic encephalopathy [
SPF grade Wistar rats, weighing 250–350 g, were obtained from Animal Center, Shengjing Hospital of China Medical University. The rats were housed in SPF facilities with a 12 h dark-light cycle in Animal Center of Shengjing Hospital of China Medical University and fed ad libitum in experiment. The animal use protocols used were approved by the Institutional Animal Care and Use Committee at China Medical University. All animal experiments were carried out in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals, as adopted by the National Institutes of Health.
Focal cerebral IR [
According to Zea Longa score standard [
The left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed but not ligated in the operation.
The rats were operated by the method of animal model, at the end of a 2-hour ischemic period, blood flow was restored by carefully removing the nylon filament.
100 mg/kg of disodium creatine phosphate (Alfa Wassermann, Inc., disodium creatine phosphate was dissolved in saline in the concentration of 50 mg/mL) was administered intravenously 30 min before operation.
These rats received the same IR, but, in contrast to the IR group, 200 mg/kg of disodium creatine phosphate was administered intravenously 30 min before operation.
These rats received the same IR, but, in contrast to the IR group, 400 mg/kg of disodium creatine phosphate was administered intravenously 30 min before operation.
We observed neurological deficits at 24 h and 72 h after occlusion, and intracranial pressure (ICP) was determined at 1 h, 2 h, 4 h, and 24 h. Apoptosis detection by TUNEL and caspase-3 immunohistochemical staining, aquaporin-4 (AQP4), and ultrastructure observation were detected at 24 h and 72 h after ischemia.
24 h and 72 h after the onset of ischemia, neurologic evaluation was performed by the same investigator, who was not aware of the group assignment. Motor behavior was evaluated using 4 tests in each rat before sacrificed [
ICP [
Brain was sampled after reperfused with sodium chloride and 4% formaldehyde, regularly embedded in paraffinum, and then sectioned at a thickness of 4
Caspase-3 immunohistochemical procedures were performed strictly according to the manufacturer’s guidelines (Nanjing Jiancheng Bio Co., Ltd). And the slides with the addition of 0.01 mmol/L PBS (containing 1 : 200 nonimmunity animal serum), instead of primary antibody, showed no response. Under the microscope, those with brown granules in cytoplasm were considered as positive cells and were counted in 5 random high-power fields (400×).
Materials of 1 mm thickness were fixed for 2 hours in 4% glutaraldehyde (pH 7.2–7.4, buffered with phosphate) and postfixed with 1% osmium tetroxide in phosphate buffer (pH 7.4) for 2 hours at 4°C. Dehydration was carried out with ethyl alcohol in a 50% concentration up to absolute. The materials were embedded in Epon resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate. Specimens were observed and photographs taken using an electron microscope. The evaluator of the tissue was blinded to the study objective and the intervention.
The protein level of AQP4 [
All data were presented as
High-dose usage of disodium creatine phosphate can cause low blood pressure. In the P3 group, 5 rats were dead from unconsciousness, which was thought to be related to low blood, pressure. 2 rats from the IR group, 1 from the P2 group and 1 from the P3 group were excluded from this study because of the development of subarachnoid hemorrhage. 1 rat from the IR group, 1 rat from the P2 group, 2 rats from the P3 group were excluded from this study because they were not scored 2–4 Zea Longa score.
24 h and 72 h after the onset of ischemia, neurologic evaluation was evaluated. There was no neurological deficit in the sham-operated group. Neurological score after ischemia reperfusion is shown in Table
Neurologic score after 24 and 72 h of ischemia after middle cerebral artery occlusion.
Group | Neurological score | |
24 h | 72 h | |
IR group | ||
P1 group | ||
P2 group | ||
P3 group |
The ICP of the five groups was shown in Figure
Intracranial pressure of five groups. There were no obviously differences of ICP among five groups in serial time points.
TUNEL and caspase-3 immunohistochemical assay were adopted to determine neuronal apoptosis. In the study, the apoptosis cells were mainly on the fridges of ischemia area. AI value and caspase-3 positive cells were significantly decreased after PCr treatment (Figures
Apoptosis index in brain slices of five groups at 24 h and 72 h. Data are presented as
Caspase-3 positive cells in brain slices of five groups at 24 h and 72 h. Data are presented as
(a) Apoptosis cells in brains by TUNEL assay after 24 h ischemia (×400). (b) Apoptosis cells in brains by TUNEL assay at 72 h ischemia (×400). (c) Apoptosis cells in brains by caspase-3 immunohistochemical stain assay after 24 h ischemia (×400). (d) Apoptosis cells in brains by caspase-3 immunohistochemical stain assay after 72 h ischemia (×400).
Clear bilayer nuclear membrane and nucleolus, rounded or oval-shaped mitochondria, were seen in neuron cells in sham-operated group. In the IR group, shrunken neucleus and aggregated chromatin toward the nuclear membrane were observed. In obviously swollen mitochondrion, cristae were disordered, cracked, or decreased, and membrane structure became incomplete or partially disappeared. In the P1 group, the nuclear membranes were clear, and the granule-like aggregation of chromatin could be seen. But organelles were also disordered. In the P2 group, slightly pycnosis, complete nucleus membrane, and less degenerated organelles could be observed. In the P3 group, the nucleus and mitochondrion changes levels were also alleviated obviously (Figure
(a) Hippocampus ultrastructural histopathologic changes in five groups at 24 h (×5000). (b) Hippocampus ultrastructural histopathologic changes in five groups at 72 h (×5000).
Immunoblot analysis showed AQP4 protein in brain homogenates from rats as bands at about 32 and 34 kDa representing the two AQP4 protein isoforms expressed in brain (Figure
Western Blot analyses of AQP4. 32 and 34 kDa representing the two AQP4 protein isoforms expressed in brain.
AQP4 relative density of five groups.
Rapid decrease of ATP has been shown after cerebral ischemia, which leads to secondary injuries. We hypothesized that supplication of an exogenous substrate before ischemia reperfusion might alleviate IR damages. Study showed that [
Studies have shown that the caspase family is the promoter and implementer of apoptosis in mammalian cells, among which, caspase-3 is the most critical downstream apoptosis protease in the caspase cascade “waterfall” [
Many investigators found the expression of caspase-3 enhanced with the increased cell apoptosis in brain, both in local and focal ischemia. The number and region of apoptosis were related to ischemia time and sensitivity of neurons. After cerebral ischemia, apoptosis cells mainly located in selective infarction area, preoptic region, corpus striatum, inner margin cortex of infarction boundary, corpus striatum, hippocampus, and olfactory tubercle [
Cerebral ischemia reperfusion leads to diverse structural changes in neural cells. Some brain regions, including the hippocampus, are more vulnerable to ischemia than others. In the experiment, we observed swelling mitochondrial, disaggregation of polyribosomes, decreased rough endoplasmic reticulum and Golgi apparatus in postischemic hippocampal neurons, which is consistent with the studies before [
Recently, the bidirectional water channel aquaporin-4 (AQP4) has been found to play an important role in brain-water homeostasis [
In the study, three doses of PCr have been adopted to prove the effect of PCr on focal cerebral ischemia-reperfusion injury. After pretreatment in three groups, animals’ IR injuries were alleviated in motor behavior, apoptosis, brain swelling, and ultrastructural pathological changes. The effects of 200 mg/kg and 400 mg/kg were not significantly different, but the side effect of PCr increased with higher dosage, especially low blood pressure. So, in the experiment, 200 mg/kg is more appropriate for treatment.
In conclusion, the study demonstrates PCr has neuroprotective effect on focal cerebral IR injury, which may relate to apoptosis. After PCr pretreatment, neurobehavioral score, infarction volume, and brain swelling are alleviated.
The authors have declared that no conflict of interests exists.
The study is supported by a grant from the Science Technology Plan of Liaoning Province (No. 2011225038).