ATP Induce Apoptosis Of Cerebral Cortex Neurons Of Embryonic Rat And Its Possible Mechanisms

Traumatic brain injury is a common disease in the department of neurology and it has a high incidence rate. Two different pathologic phases are included in the brain injury: the primary injury and the secondary injury. It is of great importance for us to lesson the secondary injury and protect the cells around the injury area, which will greatly improve the patients' prognosis. The mechanisms of the secondary injury in the injury of the central nervous system includes the edema, ischemia and hypoxia of the local tissue, the production of the free radical and peroxidation lipid, the effect of excitatory amino acids, inflammatory factor, nitrogen monoxidum and the effect of apoptosis. Recently it is proved in some research that after the injury of the central nervous system adenosine triphosphate (ATP) accumulates in the surrounding injured areas with a high concentration and then it induces the necrosis or the apoptosis of the cells. ATP is a classical material which can supply human body with energy. It is produced in mitochondrion and play its effect in the cytoplasm. In normal conditions it is in low concentration in extrocellular fluid. Wang xiaohai proved that after the spinal cord injury ATP accumulates in the surrounding areas of the injured area with a high concentration and then induce apoptosis of the spinal cord neurons. Chen ming proved that the P2X₁, P2X₄ and P2X₇ receptors are up-regulated in the surrounding injured areas after the traumatic brain injury and ATP induces the activation and apoptosis of the microglia through the receptors, which can make microglias release cytokine and promote the death of the neurons. Besides its role in the traumatic diseases, purine nucleotides can also reach high concentrations in the extracellular space under pathological conditions of hypoxia or ischemia. For example, the incubation of cortical brain slices with hypoxic medium (95%N₂,5%CO₂) for 30 minutes caused major changes in cellular morphology. Precubation with the P2 receptor antagonist PPADS prevents neuronal death, suggesting the involvement of P2 receptors. ATP also plays its role in the cause of degeneration diseases of central nervous system. Alzheimer's disease (AD) involves the progressive extracellular deposition of amyloidβ-peptide, which can damage neurons, leading to their disfunction and death. Aluminium-ATP promots the formation of amyloidβand this effect can be blocked by suramin. ATP can also modulate dopaminergic neurotransmission in the central nervous system. Exogenously applied ATP enhances the release of dopamine from the endings of neurons projecting from the substantia nigra pars compacta to the striatum via P2Y receptors. From all above we know that ATP plays an important role in the diseases of central nervous system. But the different roles of ATP on the neurons of traumatic injury have been reported and the mechanisms are still not clear. We suppose that firstly the role of ATP on the neurons is related with its concentrations and different concentration of ATP plays its role through different signal transduction pathways. On the other hand the toxic effects of ATP on the neurons can be achieved by two ways: the direct or the indirect mechanism. The direct mechanism is that ATP can effect on the receptors of neurons or glial cells and induce their death directly. The indirect mechanism includes two steps: firstly ATP effects on the glial cells and induce their activation. Then large amount of cytokines are realeased from the glial cells and promote the death of neurons. Therefore it will have important theoretical significance and clinical application value to do further research on the ATP's different roles, including its trophic effects on the growth and development of the central nervous system, its toxic or trophic role on the traumatic injury cells in central nervous system.Our work is based on the model of cultured cerebral cortex cells of embryonic rat. Different methods are used, including observing in inverted microscope, MTT technics, immunohistochemistry stain and Hoechst 33258 fluorescent stain. The dose-dependent and time-dependent toxic effect of ATP on the cultured cerebral cortex cells has been found. We also found that the activation of protein caspase-3 involved in the toxic effect of ATP on cerebral cortex cells. The protective role of suramin ( ATP' P2 antagonist) and nerve growth factor (NGF) on the injured cells induced by ATP have also been found.Our results are as follows:1. The effect of different concentrations of ATP on cultured cerebral cortex neurons and its dose-dependent toxic effect of the inducement of cell deathWe used inverted microscope to observe cells, MTT analysis and NSE immunohistochemistry stain technique to do our research of the ATP'role on the cytoactive, morphology and NSE positive neurons of cerebral cortex cells. Our research results are as follows: (1) MTT analysis shows that after treatment with different concentrations of ATP (0.1,1,5,10,100 mmol/L) 12 hours, the cytoactive is 102%,100%,73%, 59% and 34% of the normal cells. The cytoactive of 5,10,100 mmol/L ATP group is significantly decreased than the control group(P<0.05). (2) After treatment with different concentrations of ATP 24 hours, we observe cerebral cortex cell's morphology in inverted microscope, the results are as follows: The normal cells are ellipse and have large and well-stacked cell bodies. The boundary of each cell is clear. There are many synapses between different cells. The morphology of the cells in 0.1 mmol/L ATP group is almost the same as the normal cells. After treatment with 5 mmol/L ATP 24 hours, the number of cells is decreased and the cell bodies are smaller. The cells 'shape is like globe. The synapses between different cells are broken and the number of the synapses is decreased. After treatment with 10 mmol/L ATP 24 hours, the number of the cells is significantly decreased and there are clumps of dead cell debris. After treatment with 100 mmol/L ATP 24 hours, there are more dead cell debris. After treatment with different concentrations of ATP 24 hours, the number of live cells in the control group is 73.91±7.162. The other groups(0.1,5,10,100 mmol/L)are as follows:74.20±4.022, 35.45±5.410, 30.10±5.724, 19.90±1.633. The number of live cells in the 5,10,100 mmol/L group is significantly decreased than the control group(P<0.01). (3) After treatment with different concentrations of ATP 24 hours, the number of NSE positive neurons in the control group is 72.80±7.099.The other groups (0.1,5,10,100 mmol/L)are as follows: 75.00±6.667, 31.14±7.574, 26.00±6.716, 15.20±3.824. The number of NSE positive neurons in the 5,10,100 mmol/L group is significantly decreased than the control group(P<0.01).From above we conclude that ATP can induce the death of cultured cerebral cortex neurons and it is a dose-dependent effect.2. The role of different concentrations of ATP on the cultured spinal cord neuronsWe used inverted microscope to observe the cells'morphology and NSE immunohistochemistry stain technique to do our research of the ATP'role on the morphology and NSE positive neurons of spinal cord cells. Our results are as follows: (1) After treatment with different concentrations of ATP 24 hours, the number of live cells in the control group is 56.94±3.60. The other groups(0.1,10, 100 mmol/L) are as follows:58.20±3.40, 32.45±1.31, 17.89±1.63. The number of live cells in the 10,100 mmol/L group is significantly decreased than the control group(P<0.01). (2) After treatment with different concentrations of ATP 24 hours, the number of NSE positive neurons in the control group is 54.64±5.61.The other groups (0.1,10,100 mmol/L)are as follows: 55.00±26.67, 36.64±9.83, 17.28±4.89. The number of NSE positive neurons in the 10,100 mmol/L group is significantly decreased than the control group(P<0.01). From above we conclude that ATP can induce the death of cultured spinal cord cells and it is a dose-dependent effect.3. ATP induce apoptosis of cerebral cortex cells and caspase-3 plays a required role The cell'death includes two styles: apoptosis and necrosis. In order to make it clearthe nature of the cell'death induced by ATP, we did Hoechst 33258 fluorescent staining. Hoechst 33258 is a lipophilic material which can transit the cellular membrane and to bind with DNA .When excited by ultraviolet ray, glum blue fluorescence is emitted from the labeled DNA of the normal cells. When apoptosis occured ,the chromatin of the cell is condensed and the fluorescence of the labeled DNA is pyknotic and relucent. The cells which radiate pyknotic and relucent fluorescence are apoptotic cells. Our experiment results are as follows: After treatment with different concentrations of ATP 6 hours, the number of Hoechst 33258 positive cells in the control group(200 magnification) is 5.00±2.617. The other groups(5,10,100 mmol/L)are as follows: 14.00±2.927, 22.00±6.880, 26.20±9.576. The number of Hoechst 33258 positive cells in the 5,10,100 mmol/L group is significantly increased than the control group(P<0.01).The results indicate that ATP can induce apoptosis of cultured cerebral cortex cells.In order to make it clear the cellular and molecular mechanism of the apoptosis induced by ATP, we did cleaved caspase-3 immunohistochemistry stain. Apoptosis is a cascade reaction mediated by the caspase family. Firstly the factors which can cause apoptosis activate upstream caspase. Then downstream caspases are activated and followed by the hydrolysis of proteins, which can make apoptosis. Caspase-3 is a key member of the caspase family and is nomarly a target of apoptosis. Cleaved caspase-3 is the split fragment of caspase-3 after it is activated. So using immunohistochemistry stain technique to detect the quality and quantity of cleaved caspase-3 can reflect the degree of apoptosis. Our results are as follows: After treatment with different concentrations of ATP 6 hours, the number of cleaved caspase-3 positive cells in the control group is 5.00±2.190. The other groups(5,10,100 mmol/L) are as follows: 14.67±3.724, 25.00±4.000, 32.60±4.336. The number of cleaved caspase-3 positive cells in the 5,10,100 mmol/L group is significantly increased than the control group(P<0.01). From above we can conclude that the activation of caspase-3 plays a required role in the cerebral cortex cell' apoptosis induced by ATP.4. ATP P2 antagonist suramin regulates the ATP' effect of inducement of cell deathATP' receptors exist extensively in the central nervous system. Two subtypes of P2 receptors have been found: the P2X receptors and the P2Y receptors. P2X is an ionotrophic receptor and P2Y is a G protein-coupled receptor. Seven distinct P2X subtypes have been cloned from mammalian species which is P2X_1-7, while eight different mammalian P2Y subtypes have been cloned, that is P2Y_{1,2,4,6,11,12,13,14.} Suramin is a compound which is soluble in wate. Its molecular formula is C51H34N6O23Na6S6 and its molecular is 1429. It has been proved that suramin can antagonize the effect of all P2X receptors and most of P2Y receptors except P2Y4. We added both suramin and 5mmol/L ATP in cultured cerebral cortex cells and observe its effect. MTT analysis ,NSE immunohistochemistry stain were used. We also observed the cells'morphology under inverted microscope. Our results are as follows: (1) MTT analysis shows that after treatment with 5 mmol/L ATP or both 50μmmol/L suramin and 5 mmol/L ATP 12 hours, the cytoactive is 73%, 87% of the control group. There is significant difference between each group (P<0.05). (2) Before using ATP 3 minutes, we add ATP P2 antagonist suramin(50μmmol/L). After treatment with ATP 24 hours the results are as follows: the number of live cells in the control group is 73.91±7.162,the ATP group is 35.45±5.410,the suramin group is 50.30±4.945. There is significant difference between each group (P<0.01). (3) Before using ATP 3 minutes, we add ATP P2 antagonist suramin(50μmmol/L).The results of treatment with ATP 24 hours are as follows: the number of NSE positive neurons in the control group is 72.80±7.099,the ATP group is 31.14±7.574,the suramin group is 45.75±4.432. There is significant difference between each group (P<0.01). The results indicate that suramin may regulate the ATP' effect and lesson the cells' death induced by ATP.5. Nerve growth factor(NGF) can lesson the ATP' effect of inducement of cell deathNGF is a common neurotrophic factor and exits extensively in the nervous system. In the pathological conditions of traumatic brain injury, ischemical reperfusion injury and peripheral nerve injury NGF can protect neurons and lesson the apoptosis, promoting the neuronal regeneration and functional recovery. We added both NGF and 5mmol/L ATP in cultured cerebral cortex cells and did NSE immunohistochemistry stain, using MTT analysis to detect cytoactive and observed the cells'morphology under inverted microscope. Our results are as follows: (1) MTT analysis shows that after treatment with 5 mmol/L ATP or both 100 ng/mL NGF and 5 mmol/L ATP 12 hours, the cytoactive is 73% 82% of the normal cells. There is significant difference between each group (P<0.01). (2) Before using ATP 2 hours, we add NGF(100 ng/mL) in the cultured cells. After treatment with ATP 24 hours the results are as follows: the number of live cells in the control group is 73.91±7.162,the ATP group is 35.45±5.410,the NGF group is 49.90±4.748. There is significant difference between each group (P<0.01). (3) Before using ATP 2 hours, we add NGF(100 ng/mL) in the cultured cells.The results of treatment with ATP 24 hours are as follows: the number of NSE positive cells in the control group is 72.80±7.099,the ATP group is 31.14±7.574,the NGF group is 47.20±9.680. There is significant difference between each group (P<0.01). The results indicate that NGF protect cerebral cortex cells and lesson the cells' death induced by ATP.From all above we can draw such conclusions:1. Moderate and high concentrations of ATP can induce death of cultured cerebral cortex neurons and spinal cord neurons of embryonic rat. 2. Moderate and high concentrations of ATP can induce apoptosis of cultured cerebral cortex neurons of embryonic rat and caspase-3 plays a required role.3. ATP P2 receptors regulate its effect on cultured cerebral cortex neurons of embryonic rat.4. Suramin can partially antagonize the effect of ATP on cultured cerebral cortex neurons of embryonic rat.5. NGF can protect cerebral cortex neurons and can lesson the ATP' effect of inducement of cell death in cultured cerebral cortex neurons of embryonic rat.