| Objective: With high incidence,high risk of harm and low treatment,Ischemic cerebrovascular disease has become the focus of the whole society.For years,a large number of scholars have dedicated to the cerebral ischemia-reperfusion injury.However,how to change the neuronal tolerance to ischemia and ensure the normal physiological function after the blood ischemia-reperfusion has still become a diffcult problem to the study on the disease.We found that the limb ischemic preconditioning?LIP?via 3 cycles of transient occlusion?10 min?and opening of the bilateral femoral arteries immediately before global brain ischemic insult for 8 min could induce the brain ischemic tolerance by reducing the subsequent ischemia-reperfusion injury and the death of the pyramidal neurons in the hippocampal CA1 subfield of rats,and up-regulate the expression of p38 MAPK and ERK.These results confirmed the effect of limb ischemic preconditioning on brain protection.However,it is a long distance between the limbs with ischemic preconditioning and the brain suffered ischemia.How does the protection signal start and increase the expression of p38 MAPK and ERK in hippocampus of brain is not fully clear.In our further experiments,we have found that transection the femoral nerves and sciatic nerves respectively or jointly before LIP could inhibit the protection for brain ischemia and reverse the up-regulation of the expression of p38 MAPK and ERK in the hippocampus by LIP.Foreign scholars Bolte,Malhotra,et al also found that the ischemic preconditioning played a protective effect of remote organ through some neural pathway.These results indicated that the neural pathways involved in the protective effect of remote organ by LIP.In addition,theresearchers also confirmed the effect of the humoral pathway in the ischemic tolerance.For example,it was found that injection of free-radical scavenger dimethylthiourea?DMTU?reversed the protective effect on spinal cord by LIP.Montero et al reported that injection of adenosine through vein decreased the injury of myocardial by the ischemia reperfusion.Kingma et al found that using the autonomic ganglion blocker six hexamethonium and operating the nerves of extracardiac could not eliminate the blocking renal artery on the strong protection of the emyocardial.In the myocardial ischemia model of rats after limb ischemic,Lim et al observed that occlusion the femoral vein completely or transection of the femoral nerves and sciatic nerves jointly all could partly eliminate the effect of LIP on the myocardial protection.These results showed that the neural and humoral pathway participated in the protection on myocardial of LIP.In our recent studies,it has been confirmed that transection of the femoral nerves and sciatic nerves respectively or jointly inhibited the protective effect of LIP in different degree and down-regulated the expression of p38 MAPK and ERK in the hippocampal CA1 subfield.Pre-treatment with free radical scavenger DMTU or adenosine A1 receptor antagonist dipropylxanthine?DPCPX?through femoral vein before LIP partly inhibited the brain protective effect of LIP,and regulated the expression of p38 MAPK and ERK in hippocampal CA1 subfield.Moreover,injection of adenosine?Ade?through femoral vein mimiced the protective effect of LIP,and up-regulated the expression of p38 MAPK and ERK in the hippocampal CA1 subfield.These results were discussed the mechanism of the protective effect of LIP to the brain in neural and humoral mechanisms respectively.So,whether LIP could play a part in brain protection through the combination of the neural and humoral mechanisms,and affect the expression of p38 MAPK and ERK?Based on these data,the purpose of the research was to discuss the combined effect of the femoral nerve,sciatic nerve,free radical or adenosine on the brain ischemic tolerance induced by LIP,and observe the expression of p38 MAPK and ERK in the hippocampal CA1 subfield during the process.According to these studies,we will make clear the effect of the neural and humoral pathway in the brain ischemic tolerance and the up-regulation of the expression of p38 MAPK and ERK induced by LIP.It will provide a new sight on the research and development to improve the method of the neural ischemical damage tolerance and the prevention and treatment of the cerebral ischemic diseases.Method: 464 healthy male Wistar rats?weight in 250-300 g?were randomly divided into two parts:?1?Combined effect of femoral nerve,sciatic nerve and free radical on brain ischemic tolerance induced by LIP.?2?Combined effect of femoral nerve,sciatic nerve and adenosine on brain ischemic tolerance induced by LIP.1 Combined effect of femoral nerve,sciatic nerve and free radical on brain ischemic tolerance induced by LIP?1?sham group?n=16?: The rats were only exposed bilateral common carotid arteries for 8 min,without blocking the blood blow.?2?brain ischemia?BI?group?n=16?: The rats were clamped bilateral common carotid arteries for 8 min and then recovered the blood flow.?3?LIP+BI group?n=16?: The rats were occluded bilateral femoral arteries for 10 min,reperfused 10 min,repeated 3 times as limb ischemic preconditioning.Subsequently clamped bilateral common carotid arteries for 8 min and then recovered the blood flow.?4?femoral nerve transection?FNT?+LIP+BI group?n=16?: The rats were transected the femoral nerves before LIP,and then immediately clamped bilateral common carotid arteries for 8 min and then recovered the blood flow.?5?sciatic nerve transection?SNT?+LIP+BI group?n=16?: The rats were transected the sciatic nerves before LIP,and then immediately clamped bilateral common carotid arteries for 8 min.?6?FNT+SNT+LIP+BI group?n=16?: The rats were transected the femoral nerves and the sciatic nerves before LIP,and then immediately clamped bilateral common carotid arteries for 8 min.?7?DMTU+LIP+BI group?n=16?: The rats were injected DMTU?500 mg/kg?through femoral vein 1 h before LIP,and then immediately clamped bilateral common carotid arteries for 8 min.?8?DMTU+FNT+LIP+BI group?n=16?: The rats were injected DMTU?500 mg/kg?through femoral vein 1 h before transection of the femoral nerves,and then immediately given LIP and clamped bilateral common carotid arteries for 8 min.?9?DMTU+SNT+LIP+BI group?n=16?: The rats were injected DMTU?500 mg/kg?through femoral vein 1 h before transection of the sciatic nerves,and then immediately given LIP and clamped bilateral common carotid arteries for 8min.?10?DMTU+FNT+SNT+LIP+BI group?n=16?: The rats were injected DMTU?500 mg/kg?through femoral vein 1 h before transection of the femoral nerves and sciatic nerves,and then given LIP and clamped bilateral common carotid arteries for 8 min.2 Combined effect of femoral nerve,sciatic nerve and adenosine on the brain ischemica tolerance induced by LIP2.1 Combined effect of femoral nerve,sciatic nerve and adenosine A1 receptor antagonists DPCPX on the brain ischemic tolerance induced by LIPThe processing method with?1?6group is the same as 1?1?6?n=16?.?7?DPCPX+LIP+BI group?n=16?: The rats were injected DPCPX?32 nmol/kg?through femoral vein 5 min before LIP and then clamped bilateral common carotid arteries for 8 min.?8?DPCPX+FNT+LIP+BI group?n=16?: The rats were injected DPCPX?32 nmol/kg?through femoral vein 5min before transection of the femoral nerves,and then immediately given LIP and clamped bilateral common carotid arteries for 8 min.?9?DPCPX+SNT+LIP+BI group?n=16?: The rats were injected DPCPX?32 nmol/kg?through femoral vein 5 min before transection of the sciatic nerves,and then immediately given LIP and clamped bilateral common carotid arteries for 8min.?10?DPCPX+FNT+SNT+LIP+BI group?n=16?: The rats were injected DPCPX?32 nmol/kg?through femoral vein 5 min before transection of the femoral nerves and sciatic nerves,and then immediately given LIP and clamped bilateral common carotid arteries for 8 min.2.2 Combined effect of femoral nerve,sciatic nerve and adenosine on the brain ischemical tolerance induced by LIP?1?sham group?n=16?: The rats were only exposed bilateral common carotid arteries for 8 min,without blocking the blood blow.?2?BI group?n=16?: The rats were clamped bilateral common carotid arteries for 8 min and then recovered the blood flow.?3?FNT+BI group?n=16?: The rats were transected the femoral nerves.Subsequently,clamped bilateral common carotid arteries for 8 min.?4?SNT+BI group?n=16?: The rats were transected the sciatic nerves.Subsequently,clamped bilateral common carotid arteries for8 min.?5?FNT+SNT+BI group?n=16?: The rats were transected the femoral nerves and sciatic nerves.Subsequently,clamped bilateral common carotid arteries for 8 min.?6?Ade+BI group?n=16?: The rats were pre-treatment with Ade?20 nmol/kg?through femoral vein 10 min before clamping bilateral common carotid arteries for 8 min.?7?Ade+FNT+BI group?n=16?: The rats were pre-treatment with Ade?20 nmol/kg?through femoral vein 10 min before transection of the femoral nerves,and then immediately clamped bilateral common carotid arteries for 8 min.?8?Ade+SNT+BI group?n=16?: The rats were pretreatment with Ade?20 nmol/kg?through femoral vein 10 min before transection of the sciatic nerves,and then immediately clamped bilateral common carotid arteries for 8 min.?9?Ade+FNT+SNT+BI group?n=16?:The rats were pretreatment with Ade?20 nmol/kg?through femoral vein 10 min before transection of the femoral nerves and sciatic nerves,and then immediately clamped bilateral common carotid arteries for 8 min.Animals were used for two aims respectively:?1?to observe the combined effect of the femoral nerve and sciatic nerve,free radical and adenosine in the ischemic tolerance induced by LIP by thionin staining: 6 rats of each group were sacrificed by decapitation at 7 d after the sham operation or the last time of ischemia.Histological changes of the hippocampus were evaluated by histological degree?HG?and neuronal density?ND?.HG was divided into four grades referenced by Kitagawa?1990?and Kato?1991?: 0,no neuron death.I: scattered in the neuron death.II: piece of neuron death.III:almost all of the neuron death.The ND of the CA1 subfield of the hippocampus was determined by counting the number of surviving pyramidalneurons with intact cell membrane,full nucleus,and clear nucleolus within 1mm liner length of the CA1.The average number of pyramidal neurons in 3areas of the CA1 hippocampus was calculated to establish the ND.?2?to detect the expression of p38 MAPK and ERK in the hippocampal CA1 subfield of rats by immunohistochemical staining and Western blot,?1?Immunohistochemical staining: 5 animals of each group were killed by decapitation at 12 h after the last operation.According to the conventional method they were cut into setions for marking p-p38 MAPK and p-ERK antigen by p-p38 MAPK and p-ERK antibody.After colorating by DAB,the total area of positive cells and integral optical density could be calculated by microscopic image analysis system in order to quantitatively analysis the expression of p-p38 MAPK and p-ERK.?2?Western blot: The other five animals of each group were separated the hippocampus at 12 h after the last operation,then were prepared the tested samples using Western blot.According to the conventional method of Western blot they were analysed the expression of p-p38 MAPK and p-ERK quantitatively.Results:1 Combined effect of femoral nerve,sciatic nerve and free radicals on brain ischemic tolerance induced by LIP1.1 The results of thionin staining showed that the pyamidal neurons in the hippocampal CA1 subfield in sham group were arranged in order and dense for 23 layers.The outline of the neurons was intact.The cell morphology was normal,dyeing was uniformly,the nuclear membrance was clear and no significant DND was observed.HG was 0,and ND was 221.56±9.38?cells/mm,the same as followings?.However,it was observed obvious histological injury and obvious DND in the hippocampal CA1 subfield in BI group.The pyramidal neurons was changed significantly,almost all of the neuron death or missing,HG was III,and ND was 47.67±4.82.Compared with the sham group,HG was significantly increased?P<0.01?,while ND was significantly decreased?P<0.01?.In LIP+BI group there was no significant damage in the hippocampal CA1 subfield.Karyopyknosis was observed inseveral cells.The pyramidal neurons were arranged in order,and no obvious cell lossing.Compared with the BI group,HG?0I?was significantly decreased and ND?210.44±9.85?was significantly increased?P<0.01?.The histological injury of neurons in the hippocampal CA1 subfield were obviously in FNT+LIP+BI,SNT+LIP+BI,FNT+SNT+LIP+BI,DMTU+LIP+BI,DMTU+FNT+LIP+BI,DMTU+SNT+LIP+BI and DMTU+FNT+SNT+LIP+BI groups.The morphology of the cell changed,and piece or almost all of the neurons death or missing.HG was ??,while ND were 140.33±7.45,138.67±3.29,100.78±7.09,120.33±3.86,107.43±4.43,106.58± 5.64 and69.87±5.55.Compared with the LIP+BI group,HG was significantly increased?P<0.01?,and ND was significantly decreased?P<0.01?,especially in DMTU+FNT+SNT+LIP+BI group.But there were no significant difference between FNT+LIP+BI and SNT+LIP+BI groups,DMTU+FNT+LIP+BI and DMTU+SNT+LIP+BI groups?P>0.05?.These results showed that LIP obviously reduced the DND caused by brain ischemia and played a protective effect on brain.However,transection of the femoral nerves and sciatic nerves respectively or jointly and intravenous injection free radical scavenger DMTU before LIP partly inhibited the protection of LIP.Transection of the femoral nerves and sciatic nerves jointly meanwhile injection DMTU through femoral venous,the above effect was most significantly enhanced.It prompted that the neural and humoral factor free radical jointly participated in the brain ischemic tolerance induced by LIP.1.2?1?The results of immunohistochemical staining showed that there was a low level of the expression of p-p38 MAPK and p-ERK in the hippocampal CA1 subfield in sham and BI groups.The positive cell nucleus colored dark brown.The integral optical densities of p-p38 MAPK were 12.19±0.47 and12.77±0.23.The integral optical densities of p-ERK were 4.59±0.21 and4.74±0.40.The total areas of p-p38 MAPK were 5358.01 ±32.45 and5702.90±266.85.The total areas of p-ERK were 3441.77±359.49 and3485.16±283.18.In LIP+BI group,the expression of p-p38 MAPK and p-ERK were significantly increased while the positive cell nucleus stainingdeepened and coloring dark brown yellow.The integral optical densities of p-p38 MAPK and p-ERK were 68.43±0.32 and 31.12 ±0.98,respectively.The total areas of p-p38 MAPK and p-ERK were respectively 19843.38±393.69 and 15043.07±224.01.Compared with the BI group,the integral optical density and the total area of the positive cells were increased with statistical difference?P<0.01?.In FNT+LIP+BI,SNT+LIP+BI,SNT+FNT+LIP+BI,DMTU+LIP+BI,DMTU+FNT+LIP+BI,DMTU+SNT+LIP+BI and DMTU+FNT+SNT+LIP+BI groups,the expression of p-p38 MAPK and p-ERK were less,and the staining of the nuclear was shallow.The integral optical densities of p-p38 MAPK were 33.61±0.82,33.02±0.62,37.38±0.67,28.47±0.38,28.42±0.45 and 19.70±0.59.The integral optical densities of p-ERK were12.94±0.46,12.89±0.34,10.74±0.36,13.32±0.41,10.72±0.40,10.68±0.46 and8.16±0.35.The total areas of p-p38 MAPK were 9575.70±393.69,9585.04±268.08,7570.08±331.86,9577.59±244.29,7387.82±274.47,7391.85±354.45 and 5590.46±216.62.The total areas of p-ERK were 6339.90±225.00,6330.74±157.03,4494.76±167.65,6579.09±220.12,4404.48±316.31,4394.92±256.59 and 2587.94±217.21.Compared with the LIP+BI group there was a statistical difference?P<0.01?,especially in DMTU+FNT+SNT+LIP+BI group.?2?The results of Western blot showed that there were a low level of the expression of p-p38 MAPK and p-ERK in the hippocampal CA1 subfield in the sham and BI group.The IOD of p-p38 MAPK were 0.33±0.02 and0.34±0.03.The IOD of p-ERK were 0.35±0.04 and 0.33±0.03.The expression of p-p38 MAPK and p-ERK in LIP+BI group were increased significantly.The ratios of the IOD of p-p38 MAPK and p-ERK to the IOD of corresponding?-actin were 0.87±0.02 and 0.02±0.02.Compared with the BI group,there was a statistical difference?P<0.01?.In FNT+LIP+BI,SNT+LIP+BI,FNT+SNT+LIP+BI,DMTU+LIP+BI,DMTU+FNT+LIP+BI,DMTU+SNT+LIP+BI and DMTU+FNT+SNT+LIP+BI groups,the expression of p-p38 MAPK and p-ERK were significantly reduced and with a statistical difference compared with the LIP+BI group?P<0.01?,especially in DMTU+FNT+SNT+LIP+BI group.These results of immunohistochemical staining and Westernblot showed that LIP up-regulated the expression of p-p38 MAPK and p-ERK partly through femoral nerve,sciatic nerve and humoral factor free radical,and played a protective role in the brain.2 Combined effect of femoral nerve,sciatic nerve and adenosine on brain ischemic tolerance induced by LIP2.1 Combined effect of femoral nerve,sciatic nerve and the antagonist of adenosine A1 receptor DPCPX on the brain ischemic tolerance induced by LIP2.1.1 The results of thionin staining indicated that the histological injury of pyramidal neurons and the morphology of the cell in the hippocampal CA1 subfield in sham,BI,LIP+BI,FNT+LIP+BI,SNT+LIP+BI and FNT+SNT+LIP+BI groups were changed the same as 1.1.In DPCPX+LIP+BI,DPCPX+FNT+LIP+BI,DPCPX+SNT+LIP+BI and DPCPX+FNT+SNT+LIP+BI groups,the pyramidal neurons appeared obvious histological injury and the morphology of the cell changed,piece or almost all of the neurons death or missing in the hippocampal CA1 subfield.Compared with the LIP+BI group,HG?? ??was significantly increased?P<0.01?,and ND?140.78±5.71,102.22±11.25,81.22±6.48,79.44±5.31 and 58.33±2.4?was significantly decreased?P<0.01?,especially in DPCPX+FNT+SNT+LIP+BI group.These results showed that transection of the femoral nerves and sciatic nerves respectively or jointly and injection of adenosine A1 receptor antagonist DPCPX partly inhibited the protective effect.And transection of the nerves and intravenous drugs at the same time could enhance the inhibited effect by LIP,which indicated that the neural and humoral factor adenosine participated in the brain ischemia tolerance induced by LIP.2.1.1?1?The results of immunohistochemical staining showed that the trend of the expression of p-p38 MAPK and p-ERK in hippocampal CA1 subfield in sham,BI,LIP+BI,FNT+LIP+BI,SNT+LIP+BI and FNT+SNT+LIP+BI groups was the same as 1.2?1?.In DPCPX+LIP+BI,DPCPX+FNT+LIP+BI,DPCPX+SNT+LIP+BI and DPCPX+FNT+SNT+LIP+BI groups,the expression of p-p38 MAPK and p-ERK were significantly reduced and thestaining of the nuclear was shallow.The integral optical densities of p-p38 MAPK were 30.35±0.86,23.70±0.59,23.41±0.74 and 15.32±0.39.The integral optical densities of p-ERK were 16.40±0.49,11.12±0.18,11.19±0.29 and 9.45±0.30.The total areas of p-p38 MAPK were 9765.96±347.28,6668.90±354.16,6703.29±376.45 and 3528.92±395.46.The total areas of p-ERK were 4498.82±263.88,3165.54±233.04,3138.54±93.17 and 2161.92±120.90.Compared with the LIP+BI group,the above groups were all with a statistical difference?P<0.01?,especially DPCPX+FNT+SNT+LIP+BI group.?2?The results of Western blot indicated that the trend of the expression of p-p38 MAPK and p-ERK was consistent with the results of immunohistochemical staining.The above results showed that the neural and humoral factor adenosine jointly participated in the up-regulation of p-p38 MAPK and p-ERK by LIP in the hippocampal CA1 subfield of rats.2.2 Combined effect of femoral nerve,sciatic nerve and adenosine on the brain ischemic tolerance induced by LIP2.2.1 The results of thionin staining indicated that the pyramidal neurons in the hippocampal CA1 subfield of rats waere not obvious DND in sham group.HG was 0,and ND was 218.67±9.22.However,the pyramidal neurons in BI group appeared obvious histological injury and serious DND.HG was III,and ND was 48.89±8.11.Compared with the sham group,HG was significantly increased?P<0.01?,and ND was significantly decreased?P<0.01?.In FNT+BI,SNT+BI and FNT+SNT+BI groups,HG was III and ND were 47.56±5.98,46.78±6.55 and 45.44±6.81,which showed that there was not a statistical difference?P>0.05?.In Ade+BI group,the histological injury of the pyramidal neurons was significantly reduced,and only a few cells pyknosis,neurons arranged in neat and compact with no cell lossing.HG was 0I,ND was184.33±8.66.Compared with the BI group,HG was significantly decreased?P<0.01?and ND was significantly increased?P<0.01?with a significant statistical difference.In Ade+FNT+BI,Ade+SNT+BI and Ade+FNT+SNT+BI groups,it appeared obvious DND.HG was ? ?,ND were 141.44±3.8,140.33±7.6 and 120.67±4.84.Compared with the Ade+BI group,HG wasobviously increased?P<0.01?,while ND was obviously decreased?P<0.01?,especially in Ade+FNT+SNT+BI group.The above results showed that adenosine significantly reduced the DND caused by brain ischemic,and mimiced the protective effect on brain by LIP.Transection of the femoral nerves and sciatic nerves respectively or jointly partly inhibited the above effect.Furthermore,these results indicated that the neural and humoral factor adenosine participated in the brain ischemic tolerance induced by LIP.2.2.2?1?The results of immunohistochemical staining demonstrated that there was a low level of the expression of p-p38 MAPK and p-ERK in hippocampal CA1 subfield in sham,BI,FNT+BI,SNT+BI and FNT+SNT+BI groups,the positive cell nucleus staining light and coloring brown yellow.The integral optical densities of p-p38 MAPK were 12.19±0.47,12.77±0.23,12.15±0.43,12.17±0.56 and 12.11±0.12.The integral optical densities of p-ERK were 4.59±0.21,4.74±0.40,4.68±0.16,4.69±0.22 and 4.62±0.17.The total areas of p-p38 MAPK were 5358.01±32.45,5702.90±266.85,5691.00±319.26,5690.01± 380.50 and 5686.45±360.26.The total areas of p-ERK were3441.77±359.49,3485.16±283.18,3477.66±214.14,3469.28±188.70 and3466.88±252.03.In Ade+BI group,the expression of p-p38 MAPK and p-ERK were significantly increased,with the cell nuclear staining deepened and colored dark brown yellow.The integral optical density of p-p38 MAPK and p-ERK were 46.53±0.88 and 25.64±0.40.The total area of p-p38 MAPK and p-ERK were 13591.12±330.54 and 13290.42±215.10.Compared with the BI group,there was a significant statistical difference?P<0.01?,especially in Ade+FNT+SNT+BI group,which indicated that Ade could mimic the effect to the expression of p-p38 MAPK and p-ERK in the hippocampal CA1 subfield as LIP.In Ade+FNT+BI,Ade+SNT+BI and Ade+FNT+SNT+BI groups,the expression of p-p38 MAPK and p-ERK were significantly decreased with the cell nuclear staining lighter.The integral optical density of p-p38 MAPK were 35.16±0.87,35.10±0.93 and 24.86±0.85.The integral optical density of p-ERK were 18.18±0.36,18.21±0.64 and 11.11±0.19.The total area of p-p38 MAPK were 9796.71±345.65,9782.16±364.33 and6765.81±308.10.The total area of p-ERK were 11363.59±303.25,11325.14±325.21 and 9332.30±182.30.Compared with the Ade+BI group,there were significant differences?P<0.01?especially in Ade+FNT+SNT+BI group.?2?The results of Western blot indicated that the trend of the expression of p-p38 MAPK and p-ERK was consistent with the results of immunohisto-chemical staining.These results showed that neural pathway and adenosine jointly participated in the up-regulation of p-p38 MAPK and p-ERK in the hippocampal CA1 subfield of rats to brain ischemia by LIP.Conclusions:1 Transection of the femoral nerves and sciatic nerves respectively or jointly and injection of free radical scavenger DMTU or adenosine A1 receptor antagonist DPCPX through femoral vein before LIP could partly inhibit the brain protective effect of LIP.And injection of adenosine through femoral vein could play a protective effect as LIP.It demonstrated that femoral nerve,sciatic nerve,humoral factor free radical or adenosine participated in the brain ischemic tolerance induced by LIP.2 Transection of the femoral nerves and sciatic nerves respectively or jointly and injection of free radical scavenger DMTU or adenosine A1 receptor antagonist DPCPX through femoral vein before LIP could partly inhibit the up-regulation of p-p38 MAPK and p-ERK in hippocampal CA1 subfield of rats by LIP.And injection of adenosine through femoral vein could play a similar function in up-regulation of p38 MAPK and ERK as LIP.It suggested that LIP could influence the expression of p-p38 MAPK and p-ERK through the neural and humoral pathway. |
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