| Objective: To examine the neonatal Rats model with Hypoxic Ischemic Encephalopathy (HIE), treat the rats with transplantation of Neural Stem Cells (NSCs) and Granulocyte Colony-Stimulating Factor (G-CSF), observe the biological treatment effect organizationally, cellular and molecular, and provide rationale for the therapy of neonatal HIE.Methods:1. Cell preparation: Human fetal brain tissues were obtained and then cultured into NSCs spheres. Cell suspension for transplantation was made from these spheres labeled with DAPI (50μg/ml). By the use of immunofluorescence analysis, NSCs and differentiated cells (neurons and astrocytes) were respectively indentified by anti-Nestin, anti-β-Ⅲ-tubulin and anti-GFAP.2. Animal model grouping and injection of G-CSF: Postnatal day 7 Wistar rats were anesthetized. The left common carotid artery was double-ligated and cut between the ligatures. After 2～4 hours of recovery, the animals were placed in a pre-heated(39℃) and humidified chamber with a mixture of 8%O2+92%N2 for 2.5 hours. The survived rats were divided into three groups randomly: the HIE group (Group A), the single transplantation group (Group B) and the transplantation + G-CSF group (Group C). Group C received G-CSF (50μg·(kg·d)-1) 1 hour after performing operation subcutaneously once a day for five days in a row.3. Transplantation of exogenous NSCs: At the second day after the operation, NSCs were implanted into the left cerebral ventricles in rats of group B and group C. Each rat received 5μL(1×105/μL) cell suspension while group A received the same amount of normal saline as control. No immunosuppressant was given throughout the operation. 4. The observation of praxiology and anatomy: mNSS of rats was evaluated, the weight was measured, and morphous of the brain was investigated. TTC stain was done to display the infarct volume.5. Tissue processing: Brains were removed on determined times. Paraffin and frozen coronal sections were made for further processing. HE and Nissl stain were done to identify their pathological changes. Immunofluorescence analysis was used to observe the survival, migration, and differentiation of the implanted cells. The immunofluorescence images were processed by technical software for cell counting and compare the differences between group B and group C.Results:1. Cell identification: The typical NSCs spheres were obtained after cell culture, most of which were identified by detecting the NSCs'marker Nestin by immunofluorescence. After differentiation-inducing, the NSCs could differentiate into neurons and astrocytes by detecting the markerβ-Ⅲ-tubulin and GFAP.2. NSCs transplantation: Implanted cells (DAPI positive cells) survived and migrated to the injured areas. They were localized around hippocampus, cortex, brain stem and cerebellum. Implanted NSCs differentiated into different cells in different areas.3. Neurological Behavioral Measurement: All the mNSS of the rats were high post operation. Rats subjected to NSCs transplantation exhibited significantly less score than group A, moreover the score of group C were significantly less than group B, which indicated that combined therapy improved neurological behavior after HIE.4. The praxiology and anatomy: The brain weight was significantly improved in animals treated with NSCs transplantation compared with HIE in 1, 2 and 4weeks post operation (F=42.38, 121.76 and 58.22 respectively, P<0.01), more significantly in group C in 1 and 2 weeks post operation (t=2.36 and 2.63 respectively, P<0.05). TTC stain revealed that the infarct volume was significantly lower in group C than group A.5. The pathology: Confused cell arrangement, swollen neurons, and depletion of nissl's body were observed in group A, while neurons were better arranged in group B with less edema. Moreover, neurons were regular shaped and were covered with nissl's body in group C.6. TUNEL staining: Significantly decreased number of TUNEL + DAPI positive cells was observed in group C compared to group B 3 days post NSCs transplantation(181.07±14.16 VS 73.20±14.01, t=68.37, P<0.01).7. Immunofluorescence: Implanted stem cells in rats of both group B and C were detected on 1 week, 2 weeks and 4 weeks post HIE .1) The grafted cells detected by DAPI survived, migrated and generally distributed among cortex, hippocampus, striatum, and other areas. The statistical analysis revealed that DAPI positive cells were more in rats of group C than that of group B (t=14.07 and 2.5 respectively, P<0.05). While there is no significant difference between them 4 weeks post transplantation (t=1.47,P>0.05).2) Differentiated neurons could be seen by detecting DAPI+β-Ⅲ-tubulin positive cells, which located mainly at cortex and hippocampus. Statistical analysis revealed no difference of the double positive cells in the cortex 1 week post transplantation(t=1.66,P>0.05), while in the hippocampus more double positive cells were found in group C(t=3.02, P<0.01). The number in both cortex and hippocampus was significantly greater in group C than that in group B 2 weeks post transplantation (t=4.78 and 2.87 respectively, P<0.01).3) Differentiated astrocytes could be seen by DAPI+GFAP positive cells with a small amount located mainly at the cortex, striatum and white matter. Statistical analysis showed that the double positive cells were more in rats of group C than that of group B 1 week post transplantation(t=4.83,P<0.01), while no difference exited another 1week later(t=1.59, P>0.05).4) Differentiated astrocytes could be detected by DAPI+ Olig2 positive cells, which could scarcely be seen in either group.Conclusions:1. The NSCs spheres derived from human embryonic brains could be gained by culturing in media containing EGF and bFGF in vitro. Those cells possess NSCs'characteristics even when labeled with DAPI.2. Cells implanted into brains of HIE rats could survive, migrate and differentiate into neurons and astrocytes.3. Combined therapy of NSCs transplantation with G-CSF could improve brain weight, pathological renovation and promote functional recovery more efficiently in HIE rats. The survival of implanted cells was increased, apoptosis was reduced and differentiation was promoted with G-CSF treatment.
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