| PartⅠ The Study on Pramipexole-Induced Hypothermia on rats.Objective: To study different doses of Pramipexole Induced Hypothermia on rats.Methods: 1. 42 healthy male SD rats were randomly divided into seven groups with six rats per group. Each group received a single dose of pramipexole at a dose of 0, 0.125, 0.25, 0.5, 1.0, 1.5 and 2.0 mg/kg body weight by intraperitoneal injection. Changes in rat body temperature were continuously monitored for selection of the appropriate drug concentration and administration time. 2. 6 healthy male SD rats were injected of 0.3ml non-heparinized femoral artery blood into prechiasmatic cistern. 48 hours after SAH establishment, the rats were sacrificed through circulation perfusion with PBS. Monitor rat body temperature continuously, select the appropriate time to administration.Results:1. Continuous monitoring of animal body temperature showed that, compared with 0.125 mg/kg body weight group, 0.25 mg/kg body weight of pramipexole through every 6 to 12 hour intraperitoneal injection more successfully maintained rat body temperature at 34-36°C. 2.The 0.25 mg/kg body weight group had a 0% mortality rate while the 0.5, 1.0, 1.5, 2.0 mg/kg groups had various rates of death.Conclusions: The data showed that 0.25 mg/kg body weight pramipexole could safely and effectively induce hypothermia maintained at 34-36°C in normal rats.PartⅡ Effects and possible mechanisms of pramipexole-induced hypothermia on EBI after SAH.Objective: To study the effects of the dopamine receptor agonist pramipexole-induced hypothermia on EBI after SAH. Discuss the possible mechanisms.Methods: 24 healthy male SD rats were randomly divided into four groups with 6 rats in each group: normal, sham, SAH and SAH+pramipexole groups. Body temperature was continuously monitored to verify whether pramipexole was still possible of inducing hypothermia in SAH rats. SAH rats were injected of 0.3ml non-heparinized femoral artery blood into prechiasmatic cistern. 48 hours after SAH establishment, the rats were sacrificed through circulation perfusion with PBS. Brain cell apoptosis and necrosis were studied by terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) and Fluoro-Jade B(FJB) staining methods. The blood brain barrier integrity was studied by Western blot analysis of albumin in brain tissues. The Bcl-2 and Bax protein concentration, the activation of caspase-3, and the phosphorylation of PI3K/AKT/GSK3β in brain tissues were also detected by Western blot. Whether pramipexole-induced hypothermia could inhibit brain damage caused by SAH and its possible mechanism were then analyzed based on above mentioned studies.Results: 1. TUNEL and FJB staining showed that a small number of TUNEL-positive cells appeared in normal and sham groups. Compared with sham, SAH group demonstrated a significantly higher amount of TUNEL and FJB positive cells, and pramipexole-induced hypothermia could effectively reduce SAH-induced increases of TUNEL and FJB positive cells. 2. Western blot analysis of the content of albumin in brain tissues showed that brain tissues in normal and sham groups contained very low albumin indicative of blood-brain barrier integrity, and no significant difference was found between these two groups. Compared with the sham group, the albumin concentration in brain tissues in the SAH group increased significantly which suggests breach of blood-brain barrier, but pramipexole-induced hypothermia could effectively reduce the elevated levels of albumin in the brain tissues in SAH rats. 3. Western blot analysis showed that, compared with the sham group, the Bcl-2 protein levels were significantly reduced in the SAH group, while Bax protein levels and the activation of caspase-3 increased very significantly in the SAH group. Compared with the SAH group, pramipexole-induced hypothermia could effectively prevent the SAH-induced inhibition of Bcl-2, and increases of Bax and caspase3 activation. In addition, Western blot results also showed that, compared with the sham group, the SAH group demonstrated increased PI3 K phosphorylation levels, no obvious changes of phosphorylation of AKT, significantly reduced GSK3β phosphorylation levels. Compared with the SAH group, SAH + pramipexole treatment group demonstrated significantly elevated levels of phospho-PI3 K,-AKT, and-GSK3β.Conclusions: 1.Pramipexole-induced hypothermia could effectively suppress apoptosis of brain cells and ameliorate the blood-brain barrier damage caused by SAH.2. Pramipexole-induced hypothermia may inhibit apoptosis via caspase-dependent PI3K/AKT/GSK3β signaling pathways in SAH rats.Part Ⅲ Mechanisms of pramipexole-induced hypothermia on EBI after SAH.Objective: To determine whether the neuroprotective effect of pramipexole-induced hypothermia was mediated by PI3K/AKT/GSK3β signaling pathway.Methods: 30 SD rats were randomly grouped into SAH group(n = 6) and SAH + pramipexole group(n = 24). SAH + pramipexole group of 24 rats was further grouped into four groups with 6 in each group: solvent control group, PI3 K inhibitor LY294002 group, AKT inhibitor MK-2206 group and GSK3β inhibitor CHIR99021 group. SAH rats were injected of 0.3ml non-heparinized femoral artery blood into prechiasmatic cistern. 48 hours after SAH establishment, the rats were sacrificed through circulation perfusion with PBS. Whether pramipexole induces hypothermia via PI3K/AKT/GSK3β pathway was studied by TUNEL and FJB staining and Western blots, as mentioned above.Results:1. LY294002 could effectively suppress phosphorylation of PI3 K, AKT and GSK3β; MK-2206 could effectively suppress phosphorylation of AKT and GSK3β; and CHIR99021 could effectively inhibit the phosphorylation of GSK3β in SAH. 2.In the presence of any of these blockers, pramipexole-induced hypothermia could not effectively suppress caspase-3 activation induced by SAH. 3.TUNEL and FJB staining also showed that in presence of any blocker, pramipexole-induced hypothermia could not effectively suppress SAH-induced brain cell apoptosis and necrosis.Conclusions: Pramipexole-induced hypothermia, at least partially, inhibits brain damage via PI3K/AKT/GSK3β signaling pathways. |
PDF Full Text Request