| Objectives:1. To explore the neuroprotective effect of application of exogenous recombinant humanthrombopoietin (rhTPO) and Granulocyte Colony-Stimulating Factor (G-CSF) in neonatal ratswith hypoxic ischemic brain damage (HIBD), and to investigate the possibility of rhTPOtreatment and the possible mechanisms.2. To compare the differences of neuroprotective effect and side effect between differentadministrations in rhTPO and G-CSF, looking for a more precise way and provide thepossibility of laboratory evidence for the clinical treatment of neonatal hypoxic-ischemic braindamage.Methods:2507-day-old Sprague-Dawley rats (SD rat) were randomly divided into5groups (n=50in each group):①,sham operation group (C group);②,TPO treatment group;③,G-CSFtreatment group;④, HIBD1group; and⑤,HIBD2group. Left common carotid artery (CA)were ligated and cut off in the group②③④⑤, and then the models werekeeped underhypoxia environment for2hours. In sham-operation group, the median neck incision wasperformed, and left common carotid artery was isolated without ligation and hypoxia-ischemia.In TPO treatment group, the model rats were instantly given single intraperitoneal injection ofrecombinant human thrombopoietin (rhTPO)15U/10g. In G-CSF treatment group, the modelrats were instantly given subcutaneous injection of G-CSF0.5μg/10g for5straight days. Samedosage Physiological saline was given in HIBD1and HIBD2groups at same time byintraperitoneal injection or subcutaneous injection.The pathological changes and the expression of the nestin were detected after HIBDmodeling1d,2d,5d,7d,10d,14d and21d by morphological method andimmunohistochemistry method. The nestin positive cells were counted under microscope byusing Image-Pro Plus6.0. Brainstem auditory evoked potentials (BAEP) were recorded afterHIBD modeling7d,14d,21d and28d with an evoked potential recorder. Platelets werecounted afte HIBD modeling1d,7d,14d and21d in TPO, HIBD1and sham operation group.White blood cells were counted afte HIBD modeling1d,5d,14d and21d in G-CSF, HIBD2and sham operation group.Results: 1. HIBD Modeling rats had abnormal behavior with various degrees such as twitch, respiratorydisorders, disequilibrium and abnormal rotation. After modeling3days, edema and necrosisappeared at the left cerebral hemisphere in the rats of HIBD1and HIBD2group.Aftermodeling7days, the left brain atrophied and the cord-like hollow structure disorder appeared;after modeling14days, the atrophy of left cerebral is more obviously in the same groups.There are similar changes in TPO and G-CSF group, but relatively much minor, the gross andtissue morphological changes of them are similar to normal rats.2. The percentage of brain damage in TPO, G-CSF, HIBD1and HIBD2group were heavierthan group C (P<0.05) in every sampling time.The percentage of brain damage in TPO andG-CSF group were smaller than group HIBD1and HIBD2(P<0.05) in every sampling timeexcept10day and21day in TPO group.3. The expression of nestin was detected by immunohistochemistry method. The number ofnestin positive cells in TPO, G-CSF, HIBD1and HIBD2group were more than group C atevery sampling point (P<0.05). The number of nestin positive cells in G-CSF group were morethan HIBD2group at every sampling point (P<0.05).4. In14d,21d,28d, there were no significant difference (except Ⅲwave in21,28days ratsand Ⅱ wave in28days rats) of peak latency (peak latency, PL) of BAEP in TPO treatmentgroup and sham control group; In35-day-old rats, there were significant longer of PL of waveⅡ，wave Ⅲa nd waveⅣ in the TPO groups than the sham operation group (P<0.05); In14-day-old rats, there were no significant difference (except Ⅲwave in14days rats) of PL ofBAEP in G-CSF treatment group and sham control group; In21d,28d,35d, there weresignificant (except Ⅳ wave in21rats, Ⅴ wave in21days and28days rats and Ⅰwave in35days rats) longer of PL of wave Ⅰt o wave Ⅴ in the G-CSF groups than the sham operationgroup (P<0.05); In14-day-old rats, there were significant longer of PL of wave Ⅰ, waveⅢand Ⅳ wave in the HIBD1and HIBD2groups than the sham operation group (P<0.05); In21d,28d,35d, there were significant longer of PL of wave Ⅰ to wave Ⅴin the HIBD1and HIBD2groups than the sham operation group (P<0.05); In14-day-old rats, there were significantlonger of PL of wave Ⅰ,wave Ⅲ and wave Ⅴin the HIBD1groups than the TPO group(P<0.05); there were significant longer of PL of wave Ⅰ and waveⅣ in the HIBD2groupsthan the G-CSF group (P<0.05); In21d,28d,35d, there were significant longer of PL of waveⅠ to wave Ⅴ in the HIBD1or HIBD2groups than the TPO or G-CSF group (P<0.05); In28-day-old rats, there were significant longer of PL of wave Ⅱ and wave Ⅳ in the G-CSFgroup than the TPO group (P<0.05); In35-day-old rats, there were significant longer of PL of wave Ⅳ in the TPO group than the G-CSF group (P<0.05). In14d,21d,28d,35d, there weresignificant longer of most inter-peaklatency (inter-peak latency, IPL) in the TPO, G-CSF,HIBD1and HIBD2groups than the sham operation group (P<0.05), there were significantlonger (P<0.05) of IPL in HIBD1group compared with TPO treatment group, there weresignificant longer (P<0.05) of IPL in HIBD2group compared with G-CSF treatment group; In14-day-old rats, there were significant longer (P<0.05) of IPL of wave Ⅰ~Ⅲ in G-CSFtreatment group compared with TPO group; In21-day-old rats, there were significant longer(P<0.05) of IPL of wave Ⅰ~Ⅴ in G-CSF treatment group compared with TPO group; In28-day-old rats, there were significant longer (P<0.05) of IPL of wave Ⅲ~Ⅴ andwave Ⅰ~Ⅴin TPO treatment group compared with G-CSF group, there were significant longer (P<0.05)of IPL of wave Ⅰ~Ⅲ in G-CSF treatment group compared with TPO group.5. Platelets count of the TPO group was higher than HIBD1and C groups (P<0.05) only in8-day-old rats. There were no significant differences of Platelets count in TPO, HIBD1and Cgroup at other sampling point. White blood cell count of the G-CSF group and HIBD2groupwas higher than the sham operation group (P<0.05) in8,14,21-day-old rats. There were nosignificant differences of White blood cell count in these groups in28-day-old rats.Conclusions:1. In the early stage of HIBD, the administered immediately with TPO can play aneuroprotective role. It suggested that TPO could significantly improve the brain function andmorphologic change in HIBD rats; it is benefit for the development of brain.2. In the early stage of HIBD, the administered immediately with G-CSF can play aneuroprotective role. It suggested that G-CSF could mobilize and improve the neural precursorcell proliferating and differentiating to reduce the impact of degenerated and necrosis neuronsin HIBD rats.3. There are no obvious side effects by using rational TPO therapy.4. There are no obvious side effects by using rational G-CSF therapy. |
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