| Objective: Cerebral ischemic diseases, as a common disease with high morbidity, disability, mortality rate and low awareness, treatment, control rate, are harmful to human health. With the aging in our country, the serious situation of these diseases should not be ignored. In recent years, many researchers dedicate to the study of activation of endogenous protective mechanisms for improving the tolerance of neurons to the ischemic damage, in order to keep neurons which are suffered severe ischemia with normal physiological function after blood supply. The purpose of this study is to provide new control strategies for these diseases.Since 1990, a Japanese scholar named Kitagawa firstly discovered the phenomenon of brain ischemic preconditioning. Many studies confirmed that the protective effect of ischemic preconditioning in the same organ. Despite the tolerance to cerebral ischemia induced by ischemic preconditioning on central organs is a powerful endogenous protection, it is difficult to be applied to clinical work directly. As the further studies on ischemic preconditioning, many researchers discovered and confirmed the protective effect of remote ischemic preconditioning(RIPC). RIPC means that ischemic preconditioning was operated on tissues or organs except the target one, and then the tolerance to ischemia occurred on the target organ. This finding realized the ischemic preconditioning on non-vital organs withstands the ischemia-reperfusion injury on vital organs. Meanwhile, this method is relatively safe and operable.In recent studies, we found that the limb ischemic preconditioning(LIP) via 3 cycles of transient occlusion for 10 min and opening for 10 min as well of the bilateral femoral arteries immediately before global brain ischemia for 8 min could relieve the subsequent brain ischemia-reperfusion injury and reduce the death of the pyramidal neurons in the CA1 region of the hippocampus of rats. Subsequently, it has been demonstrated in our further studies that LIP could induce the tolerance to brain ischemia by up-regulating the expression of p38 MAPK, ERK, HSP 70 and Ngb in the CA1 region of the hippocampus of rats, inhibited apoptosis pathway, changed the activity of superoxide dismutase(SOD) and so on. However, the limb performed ischemic preconditioning is far away from the brain which is suffering ischemia, it is not fully clear that how the endogenous protective signal activated by LIP affect brain. In 2015, Weber NC discovered that plasma from adult male volunteers subjected to LIP could protect human endothelial cells from hypoxia-induced cell damage by reducing the activity of LDH and up-regulating the expression of HIF1α and p-ERK, which demonstrated that LIP could perform protective effect on remote organs or tissues through humoral pathway. In the further studies, researchers discovered that several humoral factors, such as free radical and adenosine, were associated to the protection of LIP. For example, in spinal cord ischemia-reperfusion injury model, intravenous injection of free-radical scavenger DMTU could reverse the protective effect on spinal cord induced by LIP. It was shown that free radical plays an important role on protective effect of LIP. In 2014, Montero discovered that heart injury following intestinal ischemic reperfusion in rats was attenuated by association of ischemic preconditioning and adenosine, which indicated that adenosine participated in the protection of remote organs. Therefore, we supposed does LIP can induce brain ischemic tolerance through humoral factors free radical or adenosine? Furthermore, does these humoral factors can up-regulate the expression of p38 MAPK and ERK in this process?The purpose of this study was: ①The effect of free radical and adenosine on tolerance to brain ischemia of rats induced by LIP. ② The effect of free radical and adenosine on the up-regulation of p38 MAPK and ERK in the CA1 region of the hippocampus of rats induced by LIP. These studies might provide sufficient experimental evidence of protective effect on brain induced by LIP and contribute to realize the mechanisms of protective effect on brain induced by LIP entirely. 1 The effect of free radical and adenosine on the tolerance to brain ischemia of rats induced by LIP 1.1 The effect of free-radical scavenger DMTU on brain ischemic tolerance of rats induced by LIPEighty Wistar rats whose bilateral vertebral arteries were occluded permanently were randomly divided into five groups: ①brain ischemic sham group(n=10): the rats were exposed bilateral common carotid arteries for 8 min, without blocking the blood blow. ② brain ischemia(BI) group(n=10): the rats were clamped bilateral common carotid arteries for 8 min and then recover the blood flow. ③ LIP + BI group(n=10): the rats were occluded bilateral femoral arteries for 10 min, three times, at 10 min intervals. Subsequently, to clamp bilateral common carotid arteries for 8 min and then recover the blood flow. ④ DMTU + LIP + BI group(n=40): the rats were injected DMTU through femoral vein 1 h before LIP and then clamp bilateral common carotid arteries for 8 min. According to the dose of DMTU, these rats were randomly divided into 0 mg/kg, 250 mg/kg, 500 mg/kg and 750 mg/kg four subgroups. ⑤ DMTU + sham group(n=10): the rats were administrated DMTU(500 mg/kg) through femoral vein and expose bilateral common carotid arteries for 8 min 1 h later.Six animals of each group were killed by decapitation at 7 d after the last operation, a 4-mm coronal brain section behind the optic chiasma was cut into slices for thionin staining in order to observe the grade of delayed neuronal death(DND). Histological changes of the hippocampus were evaluated using thionin staining by histological degree(HG) and neuronal density(ND). HG was divided into four grades: 0: no neuron death. ?: scattered single neuron death. II: mass neuron death. III: almost all neuron death. The ND of the CA1 subfield of the hippocampus was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus, and clear nucleolus within 1 mm liner length of the CA1. The average number of pyramidal neurons in 3 areas of the CA1 hippocampus was calculated to establish the ND. The blood samples of the other 4 animals of each group were collected from ventriculus dexter at 3 d after the last operation. Activity of SOD and content of MDA were measured by reagent kits according to the instructions.The thionin staining results indicated that the pyramidal neurons in the CA1 region of the hippocampus in the brain ischemic sham group and DMTU 500 + sham group were arranged in order and the outline of the neurons was intact. The cell morphology was normal, dyeing was uniformly, nuclear membrance was clear and no significant DND was observed. The HG was 0, and ND were 219.11±12.45(cells/mm, the same as following) and 216.00±9.45. Obvious DND in the CA1 region of the hippocampus was observed in BI group. The pyramidal neurons in this region were changed significantly. Compared with the brain ischemic sham group, HG(III) was significantly increased(P<0.01), while the ND(48.67±4.34) was obviously decreased(P<0.01). However, LIP + BI group and DMTU 0 + LIP + BI group did not appear obvious DND in the CA1 region, only karyopyknosis was observed in several cells. HG was 0~I, and ND were 212.44±26.35 and 209.67±23.78. In LIP + BI group, compared with BI group, HG was significantly decreased(P<0.01) and ND was significantly increased(P<0.01). Obvious histological insult was observed in hippocampal CA1 subfield in DMTU 250 + LIP + BI, DMTU 500 + LIP + BI and DMTU 750 + LIP + BI groups. The cell morphology changed and HG was I ~ II, ND were 153.44±27.20, 121.33±18.37 and 123.78±28.75. Compared with LIP + BI group, HG was significantly increased(P<0.01) and ND was significantly reduced(P<0.01). Besides, there was no significantly difference between DMTU 500 + LIP + BI and DMTU 750 + LIP + BI groups. These results shown that LIP could obviously decrease the DND of the hippocampal CA1 region induced by brain ischemia. Intravenous injection of free-radical scavenger DMTU before LIP partly inhibited the protective effect on brain induce by LIP. And the optimal dose of DMTU was 500 mg/kg. It was demonstrated that humoral factor free radical partly participated in the tolerance to brain ischemia induced by LIP. Results of SOD and MDA measurement shown that the activity of SOD was 161.73±7.86(U/ml, the same as following) and the content of MDA was 13.41±1.22( nmol/ml,the same as following) in BI group. Compared with BI group, the activity of SOD(204.02±12.43) was significantly increased(P<0.01) and the content of MDA(7.65±0.85) was significantly reduced(P<0.01) in LIP + BI group. Compared with LIP + BI group, the activity of SOD(182.30±11.33) was significantly reduced(P<0.01) and the content of MDA(11.08±1.55) was significantly increased(P<0.01) in DMTU 500 + LIP + BI group. The results indicated that free radical could participate in the tolerance to brain ischemia induced by LIP by increasing the activity of SOD and reducing the content of MDA. 1.2 The effect of adenosine A1 receptor antagonist DPCPX on brain ischemic tolerance of rats induced by LIPEighty Wistar rats whose bilateral vertebral arteries were occluded permanently were randomly divided into five groups: ①brain ischemic sham group(n=10): the rats were exposed bilateral common carotid arteries for 8 min, without blocking the blood blow. ② BI group(n=10): the rats were clamped bilateral common carotid arteries for 8 min and then recover the blood flow. ③ LIP + BI group(n=10): the rats were occluded bilateral femoral arteries for 10 min, three times, at 10 min intervals. Subsequently, to clamp bilateral common carotid arteries for 8 min and then recover the blood flow. ④ DPCPX + LIP + BI group(n=40): the rats were injected DPCPX through femoral vein 5 min before LIP and then clamp bilateral common carotid arteries for 8 min. According to the dose of DPCPX, these rats were randomly divided into 0 nmol/kg, 16 nmol/kg, 32 nmol/kg and 48 nmol/kg four subgroups. ⑤ DPCPX + sham group(n=10): the rats were injected DPCPX(32 nmol/kg) through femoral vein and expose bilateral common carotid arteries for 8 min 5 min later.Methods are same as 1.1.The thionin staining results indicated that the pyramidal neurons in the CA1 region of the hippocampus in the brain ischemic sham group and DPCPX 32 + sham group were arranged in order and no significant DND was observed. The HG was 0 and ND was 221.89±10.08 and 213.22±12.77. Obvious DND in the CA1 region of the hippocampus was observed in BI group. Compared with the brain ischemic sham group, HG(III) was significantly increased(P<0.01), while the ND(46.56±6.45) was significantly decreased(P<0.01). However, LIP + BI group and DPCPX 0 + LIP + BI group did not appear obvious DND in the CA1 region, only karyopyknosis was observed in several cells. HG was 0~I and ND were 211.56±15.59 and 213.00±14.67. LIP + BI group compared with BI group, HG was significantly decreased(P<0.01) and ND was significantly increased(P<0.01). In DPCPX 16 + LIP + BI group, the morphology of neurons in hippocampal CA1 subfield has changed. The neurons were scatteredly dead or absent. HG was I ~ II and ND was 124.56±28.05. Compared with LIP + BI group, HG was increased(P<0.01) and ND was reduced(P<0.01). Obvious histological insult was observed in hippocampal CA1 subfield in DPCPX 32 + LIP + BI group and DPCPX 48 + LIP + BI group. HG was II~III, ND were 77.00±32.25 and 71.44±28.84. Compared with LIP + BI group, HG was significantly increased(P<0.01), ND was significantly reduced(P<0.01). These results indicated that LIP could obviously decrease the DND of the hippocampal CA1 region induced by brain ischemia. Intravenous injection of adenosine A1 receptor antagonist DPCPX before LIP partly inhibited the neuroprotection on brain induce by LIP. And the optimal dose of DPCPX was 32 nmol/kg. It was demonstrated that humoral factor adenosine partly participated in the tolerance to brain ischemia induced by LIP. Results of SOD and MDA measurement shown that compared with BI group, the activity of SOD(204.02±12.43) was significantly increased(P<0.01) and the content of MDA(7.65±0.85) was significantly decreased(P<0.01) in LIP + BI group. Compared with LIP + BI group, the activity of SOD(175.07±11.18) was significantly reduced(P<0.01) and the content of MDA(12.01±1.02) was significantly increased(P<0.01) in DPCPX 32 + LIP + BI group. The results indicated that adenosine could participate in the tolerance to brain ischemia induced by LIP by increasing the activity of SOD and reducing the content of MDA. 1.3 The effect of adenosine(Ade) on brain ischemia of ratsSeventy Wistar rats whose bilateral vertebral arteries were occluded permanently were randomly divided into four groups: ①brain ischemic sham group(n=10): the rats were exposed bilateral common carotid arteries for 8 min, without blocking the blood blow. ② BI group(n=10): the rats were clamped bilateral common carotid arteries for 8 min and then recover the blood flow. ③ Ade + BI group(n=40): the rats were pretreatment with adenosine through femoral vein 10 min before clamping bilateral common carotid arteries for 8 min. According to the dose of adenosine, these rats were randomly divided into 0 nmol/kg, 10 nmol/kg, 20 nmol/kg and 40 nmol/kg four subgroups. ④ Ade + sham group(n=10): the rats were injected adenosine(20 nmol/kg) through femoral vein and expose bilateral common carotid arteries 10 min later.Methods are same as 1.1.The thionin staining results indicated that the pyramidal neurons in hippocampal CA1 subfield of rats in the brain ischemic sham group and Ade 20 + sham group were arranged in order and no significant DND was observed. Obvious DND in the CA1 region of the hippocampus was observed in BI group and Ade 0 + BI group. HG was III and ND were 46.89±7.01 and 45.89±4.89. Compared with the brain ischemic sham group, HG was significantly increased(P<0.01) in BI group, while the ND was significantly decreased(P<0.01). Ade 10 + BI group appeared obvious DND in the CA1 region. HG was I~II and ND was 145.11±29.04. Compared with BI group, HG was decreased(P<0.01) and ND was increased(P<0.01). In Ade 20 + BI group and Ade 40 + BI group, only scattered death of the neurons in hippocampal CA1 subfield was observed. HG were 0~II and 0~I, while the ND were 183.44±30.84 and 193.44±10.80. Compared with BI group, HG was significantly reduced(P<0.01) and ND was significantly increased(P<0.01). These results shown that adenosine could partly imitate the neuroprotection on brain induced by LIP. And the optimal dose of adenosine was 20 nmol/kg. It was demonstrated that humoral factor adenosine participated in the tolerance to brain ischemia induced by LIP. In BI group, the activity of SOD was 161.73±7.86, and the content of MDA was 13.41±1.22. Compared with BI group, the activity of SOD(190.14±7.41) was significantly increased(P<0.01) and the content of MDA(8.24±0.84) was significantly reduced(P<0.01) in Ade 20 + BI group. The results indicated that adenosine could be related to brain ischemia induced by LIP by up-regulating the activity of SOD and reducing the content of MDA. 2 The effect of free radical and adenosine on the up-regulation of p38 MAPK and ERK in hippocampal CA1 subfield of rats induced by LIP 2.1 The effect of free-radical scavenger DMTU on p38 MAPK and ERK in hippocampal CA1 subfield of rats induced by LIPGroups are same as 1.1.Five animals of each group were killed by decapitation at 12 h after the last operation, 1~4 mm coronal brain section behind optic chiasma was cut into slices for marking p-p38 MAPK and p-ERK antigen by p-p38 MAPK and p-ERK antibody. After colorating by DAB, total area of positive cells and integral optical density could be calculated by microscopic image analysis system in order to quantitatively analysed the expression of p-p38 MAPK and p-ERK. The other five animals of each group were killed by decapitation at 12 h after the last operation. Separated the hippocampus and prepared the sample for western blot. After gel electrophoresis and antibody incubation, electrophoretic band could be analysed by gel image analysis system in order to quantitatively analysed the expression of p-p38 MAPK and p-ERK.Immunohistochemistry results shown that there was a low level of the expression of p-p38 MAPK and p-ERK in hippocampal CA1 subfield in the brain ischemic sham, BI and DMTU 500 + sham groups. The positive cell nucleus coloring dark brown, the integral optical density of p-p38 MAPK were 12.23±1.52,12.42±1.81 and 11.95±1.30, and the integral optical density of p-ERK were 4.60±0.19, 4.75±0.23 and 4.65±0.20. The total area of p-p38 MAPK was 5366.53±926.59(μm2, the same as following) 5783.77±874.26 and 5582.38±887.95, and the total area of p-ERK were 3395.34±291.14, 3453.57±374.73 and 3610.90±210.03. The expression of p-p38 MAPK and p-ERK in the CA1 subfield in LIP + BI group and DMTU 0 + LIP + BI group significantly increased. The integral optical density of p-p38 MAPK were 68.57±6.87 and 66.37±5.03, and the integral optical density of p-ERK were 31.49±3.66 and 30.62±2.90. The total area of p-p38 MAPK were 19975.52±1718.97 and 18513.65±1591.89, and the total area of p-ERK were 15310.05±618.57 and 14944.25±545.41. The integral optical density and total area of LIP + BI group were significantly increased compared with BI group(P<0.01). Compared with LIP + BI group,the expression of p-p38 MAPK and p-ERK in hippocampal CA1 subfield in DMTU 250 + LIP + BI group, DMTU 500 + LIP + BI group and DMTU 750 + LIP + BI group significantly decreased(P<0.01). The integral optical density of p-p38 MAPK were 48.19±4.50, 36.32±3.03 and 35.29±3.18, and the integral optical density of p-ERK were 22.60±2.23, 13.27±2.01 and 13.48±2.18. The total area of p-p38 MAPK was 12924.70±1231.43, 9604.63±1077.03 and 9388.41±989.71, and the total area of p-ERK were 9562.06±355.64, 6550.33±343.35 and 6687.39±235.33. Western blot results shown that the expression of p-p38 MAPK and p-ERK in the CA1 region of hippocampus in brain ischemic sham group, BI group and DMTU 500 + sham group were very low. There was a significant increase of the expression of p-p38 MAPK and p-ERK in LIP + BI group and DMTU 0 + LIP + BI group. Compared with the IOD of BI group, the IOD of LIP + BI group significantly increased(P<0.01). The expression of p-p38 MAPK and p-ERK in DMTU 250 + LIP + BI group, DMTU 500 + LIP + BI group and DMTU 750 + LIP + BI group significantly reduced, and there was a significant statistical difference compared with LIP + BI group(P<0.01). The results of immunohistochemistry and western blot indicated that LIP up-regulated the expression of p38 MAPK and ERK in the hippocampal CA1 subfield of the brain ischemic rats partly by free radical. 2.2 The effect of adenosine A1 receptor antagonist DPCPX on p38 MAPK and ERK in hippocampal CA1 subfield of rats induced by LIPGroups are same as 1.2 and methods are same as 2.1.Immunohistochemistry results showed that there was a low level of the expression of p-p38 MAPK and p-ERK in hippocampal CA1 subfield in the brain ischemic sham, BI and DPCPX 32 + sham groups, and the positive cell nucleus coloring dark brown, the integral optical density of p-p38 MAPK were 12.23±1.52, 12.42±1.81 and 12.96±1.09, and the integral optical density of p-ERK were 4.60±0.19, 4.75±0.23 and 4.63±0.19. The total area of p-p38 MAPK was 5366.53±926.59, 5783.77±874.26 and 5850.75±722.59, and the total area of p-ERK was 3395.34±291.14, 3453.57±374.73 and 3322.09±291.01. The expression of p-p38 MAPK and p-ERK in the CA1 subfield in LIP + BI group and DPCPX 0 + LIP + BI group significantly increased. The integral optical density of p-p38 MAPK were 68.57±6.87 and 65.67±5.69, and the integral optical density of p-ERK were 31.49±3.66 and 30.88±2.43. The total area of p-p38 MAPK were 19975.52±1718.97 and 18239.42±1594.06, and the total area of p-ERK were 15310.05±618.57 and 16071.61±689.89. The integral optical density and total area of LIP + BI group were significantly increased compared with the BI group(P < 0.01). Compared with the LIP + BI group,the expression of p-p38 MAPK and p-ERK in hippocampal CA1 subfield in DPCPX 16 + LIP + BI group, DPCPX 32 + LIP + BI group and DPCPX 48 + LIP + BI group significantly decreased(P<0.01). The integral optical density of p-p38 MAPK were 38.74±4.02, 28.05±2.71 and 27.40±2.20, and the integral optical density of p-ERK were 23.49±1.84, 16.24±1.32 and 16.88±1.43. The total area of p-p38 MAPK were 12340.18±1056.76, 8963.94±935.41 and 9396.58±850.62, and the total area of p-ERK were 9417.67±345.75, 7826.61±233.91 and 8013.11±252.23. Western blot results indicated that the expression of p-p38 MAPK and p-ERK was consistent with the results of immunohistochemistry. The results of immunohistochemistry and western blot indicated that LIP up-regulated the expression of p38 MAPK and ERK in the hippocampal CA1 subfield of the brain ischemic rats partly by adenosine. 2.3 The effect of adenosine on p38 MAPK and ERK in hippocampal CA1 subfield of the brain ischemic ratsGroups are same as 1.3 and methods are same as 2.1.Immunohistochemistry results demonstrated that there was a low level of the expression of p-p38 MAPK and p-ERK in hippocampal CA1 subfield in the brain ischemic sham group, BI group, Ade 0 + BI group and Ade 20 + sham group, and the positive cell nucleus coloring dark brown. The integral optical density of p-p38 MAPK were 12.23±1.52, 12.42±1.81, 13.02±1.18 and 14.54±1.34, and the integral optical density of p-ERK were 4.60±0.19, 4.75±0.23, 4.66±0.20 and 4.59±0.31. The total area of p-p38 MAPK were 5366.53±926.59, 5783.77±874.26, 5529.65±766.16 and 5272.40±742.64, and the total area of p-ERK were 3395.34±291.14, 3453.57±374.73, 3635.09±255.83 and 3486.83±310.10. The expression of p-p38 MAPK and p-ERK in the CA1 subfield in Ade 10 + BI group, Ade 20 + BI group and Ade 40 + BI group significantly increased. The integral optical density of p-p38 MAPK were 23.09±1.46, 41.61±3.22 and 39.57±5.02, and the integral optical density of p-ERK were 12.61±1.67, 25.60±2.06 and 24.54±1.59. The total area of p-p38 MAPK were 9078.42±909.58, 14711.33±1226.75 and 14549.98±1129.92, and the total area of p-ERK were 8551.07±368.26, 13350.40±651.58 and 12869.38±734.89, which were significantly increased compared with the BI group(P<0.01). Western blot results showed that the expression of p-p38 MAPK and p-ERK in the CA1 region of hippocampus in brain ischemic sham group, BI group, Ade 0 + BI group, and Ade 20 + sham group were very low. Compared with BI group, there was a significant increase of the expression of p-p38 MAPK and p-ERK in Ade 10 + BI, Ade 20 + BI and Ade 40 + BI groups. The results of immunohistochemistry and western blot indicated that adenosine could up-regulate the expression of p38 MAPK and ERK in the hippocampal CA1 subfield of the rats to induce tolerance to brain ischemia.Conclusions:1 Injection of free-radical scavenger DMTU or adenosine A1 receptor antagonist DPCPX through femoral vein before LIP could partly reverse the protective effect on brain induced by LIP. Administration of adenosine could partly imitate the protective effect on brain induced by LIP. It suggested that humoral factors free radical and adenosine participated in the protective effect on brain induced by LIP.2 Injection of free-radical scavenger DMTU or adenosine A1 receptor antagonist DPCPX through femoral vein before LIP could partly inhibit the up-regulation of the expression of p38 MAPK and ERK in hippocampal CA1 subfield of brain ischemic rats induced by LIP. Administration of adenosine could up-regulate the expression of p38 MAPK and ERK in hippocampal CA1 subfield of brain ischemic rats. It demonstrated that LIP could up-regulate the expression of p38 MAPK and ERK in hippocampal CA1 subfield of brain ischemic rats through humoral factors free radical and adenosine. |
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