Study On STAT3 Activation Following Cerebral Ischemia/reperfusion And Its Role In The Neuroprotection Induced By HBO Preconditioning

Background: Ischemic cerebrovascular diseases often lead to neurological deficits which seriously influence the quality of life and even threaten the survival of patients.With the progress of studies on the molecular mechanisms and histopathology of cerebral ischemia/reperfusion injury, a number of measures and drugs have been proved to protect neurons from ischemia/reperfusion injury. The sheer variety of stimuli capable of inducing an ischemia-resistant phenotype in the brain indicates that the signaling pathways activated by these different triggers converge downstream on some common, fundamental mechanisms that ultimately account for the protection. JAK-STAT pathway can be quickly activated which transducers the extracellular signals into cells, in which STAT3 is an important member. This study was designed to explore the activation of STAT3 after cerebral ischemia/reperfusion, and to investigate the role of STAT3 activation on the neuroprotection induced by HBO preconditioning. This study was divided into two parts, in part one the possible mechanism and the effects of STAT3 activation after cerebral ischemia/reperfusion were investigated while the role of STAT3 activation in neuroprotection induced by HBO preconditioning was investigated in part two.Part 1 The possible mechanism and the effects of STAT3 activation after cerebral ischemia/reperfusionMethods1. The STAT3 phosphorylation after cerebral ischemia/repersuion 18 male SD rats, weighting 280-320g, were randomized into 2 groups (n=9 each): sham group and MCAO group, in which the focal cerebral ischemia was induced by middle cerebral artery occlusion with 3-0 nylon monofilament for 120min. At 30min, 2h and 24h after reperfusion, the STAT3 activation was examined by western blotting.2. The distribution characteristics and cellular localization of pSTAT3 after cerebral ischemia/reperfusion18 male SD rats were divided into two groups (n=9 each) as described in 1. At 30min, 2h and 24h after reperfusion, immunohistochemistry staining was used to detect the distribution characteristics of pSTAT3, and GFAP/pSTAT3, NeuN/pSTAT3 and OX-42/pSTAT3 double-staining immunohistochemistry were performed to detect the pSTAT3 positive cells.3. Topographical relation between the distribution of pSTAT3 immunoreactive cells and angioarchitecture16 male SD rats were divided into two groups as described in 1 (n=8 each). Twenty-four hours after reperfusion, rats in each group were perfused by using tannic acid-ferric chloride method (TA-Fe) to stain the microvessel in the brain and immunocytochemistry technique was used as well to demonstrate the pSTAT3- immunoreactive cells in the slices.4. The effect of different ischemic time on STAT3 activation12 male SD rats were divided into 4 groups. Rats in I30/R120 group, I60/R120 and I120/R120 groups were subjected to 30min, 60min and 120min ischemia and reperfusion for 120min respectively. Rats in I120/R0 group were subjected to 120min MCAO but reperfusion was omitted. Then western blotting was used to detect the difference in STAT3 phosphorylation in these groups.5. The effect of ischemic or reperfusion injury on STAT3 activation18 male SD rats were divided into 6 groups. In I150 group, I180 and I240 groups, rats were subjected to 150min, 180min and 240min ischemia respectively, while rats in I30/R120 group, I60/R120 and I120/R120 groups were subjected to 30min, 60min and 120min ischemia and 120min reperfusion respectively. Then western blotting was used to detect the difference in STAT3 phosphorylation in these groups.6. The relationship of STAT3 activation and ROS15 male SD rats were divided into 5 groups: Control group, rats in NS group received intraperitoneally normal saline, and rats in DMTU1, DMTU2 and DMTU3 groups received intraperitoneally 5ml/kg 5%, 10% and 20% DMTU respectively 1h before MCAO. Then western blotting was used to detect the difference in STAT3 phosphorylation in these groups.7. The effect of tyrosine phosphorylation inhibitor AG490 on the focal cerebral ischemic injury.7.1 The dose-effect relation of AG490 for STAT3 phosphorylation inhibition 15 male SD rats were divided into 5 groups (n=3 each): Control group, animals in DMSO group were intracerebroventricularly infused with 3%DMSO, and rats in AG490-1, AG490-2 and AG490-3 groups received intracerebroventricularly 10μL 250μM,500μM and 1000μM AG490 respectively 20min before MCAO. Then western blotting was used to detect the difference in STAT3 phosphorylation in these groups, thus the optimal dose for subsequent experiments can be determined.7.2 The effect of AG490 on focal cerebral ischemic injury.30 male SD rats were randomly divided into 3 groups (n=10 each): Control, DMSO and AG490 groups. Rats in DMSO and AG490 groups were intracerebroventricularly infused with 3%DMSO and AG490 respectively 20min before MCAO. The Neurological deficit scores (NDS) were assessed at 24, 48 and 72h after reperfusion. Then at the 72h after reperfusion, the animals were decapitated and brain infarct volumes were evaluated with 2% 2, 3, 5-triphenytetrazolium chloride (TTC) staining.Results1. The expression, distribution characteristics and cell localization of pSTAT3 after cerebral ischemia/reperfusion and the relationship between pSTAT3 and cerebral vessels.We did not detected phosphorylated STAT3 protein band in sham-operated animals by western blotting while the pSTAT3 in MCAO group at different designed time points after reperfusion increased dramatically. The pSTAT3 was first detected at 30min of reperfusion, and peaked at 24h of reperfusion. This indicates within 24h after reperfusion, STAT3 phosphorylation was increased with a prolonged reperfusion time.No pSTAT3 positive cell was detected by immunohistochemistry in sham-operated animals at 30min, 2h and 24h after reperfusion. But a large number of pSTAT3 positive cells were detected in the animals subjected to MCAO at 30min after reperfusion. The STAT3 activation maintained to 24h after reperfusion. However, there was no significant difference at difference time points after reperfusion. It was also found that the pSTAT3 expression was more significant in some regions when compares to others in brain.The pSTAT3 was mainly located in the GFAP positive astrocytes and NeuN positive neurons within 24h after reperfusion, but pSTAT3 were not deteced in OX-42 positive microglia.At 24h after reperfusion, the distribution pattern of pSTAT3 immunoreactive cells in brain especially in the hypothalamus and supraoptic nucleus was coincidence with the density of microvessel in the region. More pSTAT3-immunoreactive cells were seen in the area where the microvessels were abundant. Furthermore, many positive cells located in close vicinity to the vessels.2. Effects of ischemic time, ischemia or reperfusion and ROS on STAT3 activationSTAT3 was phosphorylated in the animals subjected to 120min MCAO when reperfusion was omitted, which was significant lower when compared to those subjected to 30min, 60min and 120min ischemia and 120min reperfusion in the ipsilateral cortex and hippocampus. But there was no significant difference among I30/R120,I60/R120 and I120/R120 groups. This indicates that within 120min, different ischemic time can not change the extent of STAT3 phosphorylation. The level of pSTAT3 both in the ipsilateral cortex and hippocampus in I30/R120 , I60/R120 and I120/R120 groups significantly increased when compared to I150,I180 and I240 groups respectively. This indicates at the same time course, ischemia/reperfusion is more powerful in increasing STAT3 phosphorylation than ishcmia.There was no significant difference of the pSTAT3 level in NS and Control groups. But the level of pSTAT3 in the groups of different DMTU dose was decreased dramatically when compared to Control group. The pSTAT3 level in 20% DMTU group was lowest. We demonstrated that DMTU can decrease the STAT3 phosphorylation in a dose dependent manner, which indicates the importance of ROS in STAT3 activation.3. The effect of tyrosine phosphorylation inhibitor AG490 on focal cerebral ischemic injury.1000μM 10μL AG490 was determined to be used in the subsequent studies.The NDS in AG490 group at 24, 48 and 72h after reperfusion was significantly higher that that in the Control and DMSO groups. But there were no significant difference between the control and DMSO groups.The infarct volume at 72h after reperfusion in AG490 was significant smaller than that in the Control and DMSO groups. But there were no significant difference between the control and DMSO groups. These results indicate STAT3 activation aggravates cerebral tissue injury. Part 2 Role of STAT3 activation in the neuroprotection induced by HBO preconditioningMethods1. Change of STAT3 phosphorylation after repeated HBO preconditioning 72 Male SD rats were divided into two groups: Control and HBO groups in which animals were preconditioned with normal air or hyperbaric oxygenation (100%O2, 2.5ATA, 1h/d, 5d). 36 animals were killed at 30min, 2h and 24h after the last precondition treatment. The other 36 animals were subjected to MCAO 24h after last precondition for 2h and were killed at 30min, 2h and 24h after reperfusion. Immunohistochemistry and western blotting were use to detect the difference of pSTAT3 expression between the two groups at each time points.2. The effect of tyrosine phosphorylation inhibitor AG490 on the neuroprotection induced by HBO preconditioning40 SD male SD rats were randomly divided into 4 groups (n=10 each). Animals in Control group were subjected to cerebral ischemia for 2h after five-day normal air preconditioning, while rats in HBO group were preconditioned with HBO and were subjected to cerebral ischemia for 2h at 24h after the last HBO treatment, animals in the HBO+DMSO and HBO+AG490groups were preconditioned by HBO for 5 days and intracerebroventricularly infused with 3%DMSO and AG490 respectively 20min before MCAO. The Neurological deficit scores (NDS) were assessed at 24, 48 and 72h after reperfusion. Then at the 72h after reperfusion, the animals were decapitated and brain infarct volumes were evaluated with 2% 2, 3, 5-triphenytetrazolium chloride (TTC) staining. Results1. Change of STAT3 phosphorylation after repeated HBO preconditioningThere was no STAT3 activation both in control group and HBO group at each time point before the occurrence of ischemia. However, the STAT3 activation was significant decreased in HBO group compared to Control group after cerebral ischemia/reperfusion.2. The effect of AG490 on the neuroprotection induced by HBO preconditioningThe NDS in HBO, HBO+DMSO and HBO+AG490 groups at 24, 48 and 72h after reperfusion were significantly higher that that in the Control group. But there were no significant difference among the HBO, HBO+DMSO and HBO+AG490 groups.The infarct volumes at 72h after reperfusion in HBO, HBO+DMSO and HBO+AG490 groups were significant smaller than that in the Control group. But there were no significant difference among the HBO, HBO+DMSO and HBO+AG490 groups.Conclusions1. There was significant STAT3 activation after cerebral ischemia/reperfusion which mainly located in astrocytes and neurons, and the characteristic of pSTAT3 distribution was related to the angioarchitecture.2. STAT3 activation mainly occurred in reperfusion phase, and is associated with the robust production of ROS in this phase.3. Inhibition of STAT3 activation protects cerebral cells against ischemic injury. 4. HBO preconditioning reduces STAT3 activation, thus induces neuroprotection.