Experimental Effect Of Granulocyte Colony-Stimulating Factor On The Treatment Of Neonatal Rats With Severe Hypoxic-Ischemic Encephalopathy

Objective:To examine the treatment of granulocyte colony-stimulating factor by subcutaneous injection on the neonatal rat model with severe hypoxic ischemic brain damage,and the assessment of their recovery of injured neural cell organically and cellularly,and by the neurological score.And to discuss the mechanism,and to provide theoretical foundation for the therapy of neonatal hypoxic-ischemic encephalopathy.Methods:1.Establishment and identification of neonatal rat model with severe HIBD:The neonatal Sprague-Dawley rat aged 7 days,after anesthetized their left common carotid arteries double-ligatured and cut between the ligatures and then recovery of 2～3 hours,was exposed to 150 minutes of hypoxia(8%O₂+92%N₂) by placing them in a chamber partially submerged in water(39℃).And their extent of damage was identified.2.Determination of the time window of G-CSF treatment in HIE:SD rat aged 7 days received subcutaneous injection of G-CSF.And the CD34⁺/WBC of peripheral blood was analyzed by flow cytometer.3.Treatment of rat with HIE by G-CSF:The HIBD model rat was randomly divided into 3 groups:the double phasic group(HG₂),the single phasic group(HG₁) and the HIBD group(H).G-CSF was injected according to the time window in group A,B,C.Every pup received intraperitoneal injection of accumulated BrdU everyday until executed.4.Therapeutic effect:To investigate the praxiology by mNSS,and the anatomy of the brain at the definitive time.The infarct volume of brain was measured and the apoptosis and regeneration of neural precursor cell,neuron and glial cell were observed. Results:1 Model identification:All rats performed abnormally in behavior after HIBD.Decreased brain weight and infarct volume were observed.HE and Nissl staining showed swollen neuron,confused cell arrangement,unsharp image of nucleolus and nissl's bodies. Cortex and hippocampus were the main impaired area.2 Neurological Behavior:The assessment of mNSS in all rats was higher after operation. The score of rats in group HG₁ and HG₂ was significantly lower than comparison group after 14-day G-CSF treatment.Moreover the score of rats in HG₂ group was significantly lower than that of HG₁ group.3 Brain weight:The brain weight of animal was significantly increased after G-CSF treatment,furthermore HG₂ group showed better effect than HG₁ group.TTC staining showed a decreased infarct volume in G-CSF-treated animal compared with the control group.The group HG₂ was more effective than HG₁.4 Neural cell:HE and Nissl staining were used to explore the neural cell.The result revealed that neuron in rat treated by G-CSF was line up in order.The recovery of vacuolation,neuron loss were more obvious in HG₂ group than that of HG₁ group.5 The regeneration of neural cell:The apoptotic cell reduced by 74.53%after G-CSF treatment.Wherever the regenerative NPC,neuron and neurogliocyte were increased obviously after G-CSF treatment.Conclusions:1 Using the modified method we found all the HIBD model rats having obvious change in praxiology,the extent of brain damage was more serious and the mortality was higher than that of the classical method.It was illustrated that the modified method was feasible and can provide a reference for the use of peer.2 The administration of double phasic G-CSF to SD rat could effectively maintain the peripheral blood haemopoietic stem cell at a high level stablely for a long time, providing a large number of HSCs migrating to central nervous system and helping improve the neuroprotection of G-CSF. G-CSF play a significant neuroprotection in neonatal rat with severe HIE,moreover, the effectiveness of the double phasic group was more effective than that of the single one and HIBD.