Neuronal Protection Induced By Granulocyte Colony-stimulating Factor In The Lithium-pilocarpine-induced Se Rat Model

BackgroundEpilepsy is a common, chronic and reproductive neurological disorder, affecting up to 3% of the population of all ages, and is characterized by recurrent, unprovoked seizures. Status epilepticus (SE) can increase the risk of subsequent seizures and cause profound cerebral damages, accompanied by a characteristic pattern of neuronal cell loss that occurs preferentially in the hippocampus. Such injury may arouse impairments on cognitive functions (including study and memory), epilepsy or even death, which latter affects the patients'life quality and social adjustment and become an important factor inducing the reproductive epilepsy. In spite of the introduction of new pharmacological therapies and the increased success rate of surgical treatments for epilepsy, a substantial number of patients either did not get seizure-free or they endured major adverse events. Therefore, it is an urgent task to explore the exact mechanism of apoptosis induced by seizures and find effective therapeutic reagents. In recent years, accumulating evidences have proved that G-CSF, an endogenous hematopoietic factor, mediates neuroprotection with multiple functions in neuronal protection models. In this study, we examined the effect of G-CSF treatment on neuronal death in hippocampus and cognitive impairment during lithium-pilocarpine induced SE. AIMTo explore neuronal protection and its possible mechanism of G-CSF in the lithium-pilocarpine-induced SE rat model and supply new ideas for epilepsy treatment.Methods1. Rats were randomly assigned to four groups and used to test several indicators at the scheduled time: the control rats treated with saline, rats administered with G-CSF alone (G-CSF), rats administered with lithium-pilocarpine (L-P) and rats administered with both G-CSF and lithium-pilocarpine (G+L-P). G-CSF was given to the rats subcutaneously for three times: 12 h before pilocarpine treatment, right after diazepam treatment and 24h after diazepam treatment.2. Epilepsy:lithium-pilocarpine-induced SE rat model.3. Morris water maze: Rats were submitted to Morris water maze for acquisition phase assays from the 15th days on after the treatments and to observe the improvement on the learning and memory induced by G-CSF. Nissl staining was applied to explore the histological changes on hippocampus region.4. Brain tissues of tested rats were sliced on the 3rd and 5th days after the treatment. The hippocampus neuron apoptosis and the apoptosis-related molecules expression were determined with TUNEL and IHC.5. The tested rats were sacrificed on the 6th, 24th and 48th hours after SE induction. The hippocampus tissues were valued on AKT and ERK signaling pathways with western blot.6. Hippocampus neurons were isolated from the fetal rats and cultured in vitro. Exogenous G-CSF was added and the roles of G-CSF secreted by neuron were observed.Results: 1. The rats treated with L-P or G+L-P showed impaired performance compared with the other two groups on the third and the fourth day for the significant difference was observed between these groups. EL time spent by L-P and G+L-P was significantly different on the third and the fourth day. On the fifth day, rats of L-P group spent significantly more time than the control and G-CSF treated groups. However, no obvious difference was found between the L-P and G+L-P groups.2. Nissl staining of 14th days after SE presented that Nissl positive cells on the CA1 region of L-P treated group were significantly less than the control group. Positive cells in the G+L-P group were obviously decreased than the normal group but elevated than the L-P treated group.3. TUNEL positive cells on the CA1 region of G+L-P treated group were significantly reduced than the L-P treated group on the 3rd and 5th days after the SE.4. Bax positive cells on the CA1 region of G+L-P treated group were significantly reduced than the L-P treated group on the 3rd and 5th days after the SE.5. Bcl-xl expression was obviously reduced in the L-P treated group and the reduction degree of of G+L-P treated group were relatively less on the 3rd and 5th days after the SE.6. Caspase-3 positive cells on the CA1 region of G+L-P treated group were significantly reduced than the L-P treated group on the 3rd days after the SE but the phenomenon was absent on the 5th day after SE.7. The results of western blot revealed that G-CSF treatment prolonged the expression of p-AKT and reduced PTEN expression. No difference of ERK signaling pathway was observed between the G+L-P and L-P groups. 8. Hippocampus neuron could produce G-CSF when being stimulated by exogenous G-CSF.Conclusion:1. G-CSF enhance the learning and memory ability of SE rats.2. G-CSF exerted anti-apoptosis function on rat neuron of SE models. The function were closely related with the balance between apoptotic molecules.3. AKT signaling pathway played important roles in anti-apoptosis function in SE models and the ERK function was not clear.4. Hippocampus neurons secreted G-CSF in an autocrine way, which extended its anti-apoptotic time course.