Mri Tracer Study Of Facial Nerve Injury In The Rat Brain Transplanted Spio-labeled Gfp Transgenic Fetal Mouse Neural Stem Cells

Experiment one:Establishment of the model of facial nerve injury on ratObjective Animal experiment is the basis of the study on facial nerve diseases.It all needs to construct an ideal model for the research on changes of pathology and physiology after facial nerve injury,on process of nerve regeneration and on etiological factor and treatment of facial nerve diseases.This experiment aims to establish a model of facial nerve injury on rat in order to set up the foundation for the study of transplanting NSCs into rat brain after facial nerve injury.Methods 12 SD rats were randomly divided into 2 groups:1 week and 3 week after model induction.Then the right facial nerve was softly drug out about 3 mm from stylomastoid foramen before cut off at the root of stylomastoid foramen.1 week and 3 week following model induction,rats were killed respectively.Then paraffin sections of brain tissues were prepared.HE,TB and TUNEL staining were made to observe the neuronal apoptosis of facial nucleus on both sides.Results There were no obvious neuronal apoptosis of facial nucleus on the healthy side and injured side of 1 week group.But on the injured side of 3 week group there was remarkable neuronal apoptosis of facial nucleus.Conclusion The model made in this experiment induces remarkable neuronal apoptosis of facial nucleus successfully and have good reproducibility.So it settles good foundation for the study of transplanting NSCs into rat brain after facial nerve injury.Experiment two:Isolation,culture and identification of NSCs derived from GFP transgenic embryonic miceObjective To prepare donor cells for the study of transplanting NSCs into rat brain after facial nerve injury by isolating,culturing and identifying NSCs derived from GFP transgenic embryonic mice.Methodsâ‘ Hippocampus isolated from GFP transgenic embryonic mice was blown mechanically and collected in centrifuge tube.Serum-free medium containing 1% B27,20 ng/ ml bFGF and 20 ng/ ml EGF was added into the tube after centrifugalization to suspend the cells.After the cells were dissociated into single cell, the density was adjusted to 2×10⁵ cells/ml.Then cells were inoculated into culture plate and cultured in 37℃,5%CO₂ incubator.About 5 to 7 days the cells proliferated and formed neurospheres.Then cells were passage by isolating mechanically.After that,cells were passage every 5 to 7 days.Cells growth and GFP expression were observed with invert phase-contrast light microscope and fluorescence microscope respectively.â‘¡Primary and passage cells were identified by anti-nestin immunocytochemical staining.â‘¢Cells morph and GFP expression were observed with fluorescence microscope after differentiation induced by fetal bovine serum. Identification of neuron,astrocyte and oligodendrocytes were performed by NeuN, GFAP and O₄ immunocytochemical staining respectively.Resultsâ‘ Cells isolated from GFP transgenic embryonic mice were grown in the matter of suspensions and formed typical neurospheres after culturing 48 hours.They were able to serial passage and form clones in vitro.Meanwhile the primary and passage cells both showed GFP positive.â‘¡Immunocytochemical method showed the primary and passage cells were nestin positive.â‘¢Cells induced by FBS differentiated and immunocytochemical method showed they were NeuN,GFAP and O₄ positive respectively.The differentiated cells also showed GFP positive.Conclusion The cells isolated from GFP transgenic embryonic mice are nestin positive and possess the abilities of proliferation,self-renew and multipotency of differentiating into neuron,astrocyte and oligodendrocyte which are believed to be the main characteristics of NSCs.They also stably express GFP.So the cells isolated from GFP transgenic embryonic mice could be considered as one of the ideal donor cells in experimental research on NSCs transplantation therapy in future. Experiment three:SPIO labeling and in vitro MRI of NSCs derived from GFP transgenic embryonic miceObjective To explore the feasibility of magnetic labeling of NSCs with SPIO and to investigate the possibility of in vitro detection of magnetically labeled NSCs with a clinical 1.5 T MR.Methodsâ‘ SPIO was mixed with PLL to obtain a complex of SPIO-PLL,in which the ultimate concentration of Fe and PLL was 25ug/ml and 0.75ug/ml respectively. Then NSCs were cultured in the culture media containing SPIO-PLL in 37℃,5%CO₂ incubator for about 48 hours to label NSCs with SPIO.â‘¡Morph and expression of GFP of labeled NSCs were observed with invert phase-contrast light microscope and fluorescence microscope respectively.â‘¢Prussian blue staining was performed for demonstrating intracytoplastic nanoparticles at 24 hours and 5 days after cells induced differentiation respectively.â‘£Trypan blue exclusion test for cell viability were performed at 2,4,6 days after labeling respectively.⑤MTT test for cell proliferation were performed at 2,4,6,8 days after labeling respectively.â‘¥Cell cycle,cellular apoptosis and necrosis of labeled NSCs were studied by flow cytometry.Meanwhile cellular apoptosis were also detected by TUNEL staining.⑦After differentiation induced by FBS,identification of neuron,astrocyte and oligodendrocytes were performed by NeuN,GFAP and O₄ immunocytochemical staining respectively.⑧1×10⁶,1×10⁵,1×10⁴ and 1×10³ cells were respectively suspended in 0.5 ml gelatin (1%) for in vitro MRI at 1.5 T MR imaging system.A tube contain gelatin used as negative control.Resultsâ‘ There were no significantly difference in cell morph and GFP expression between labeled and unlabeled cells.But the colour of labeled cells was buffy under light microscope.â‘¡Numerous intracytoplastic iron particles were stained with Prussian blue staining in the SPIO-PLL labeled NSCs and differentiated cells.But there were no iron particles in unlabeled cells.â‘¢There were no significantly difference in Trypan blue exclusion test for cell viability between labeled and unlabeled cells at 2,4,6 days after labeling.â‘£There were no significantly difference in MTT test for cell proliferation between labeled and unlabeled cells at 2,4,6,8 days after labeling.⑤There were no significantly difference in cell cycle,cellular apoptosis and necrosis between labeled and unlabeled cells at 2 days after labeling. Cellular apoptosis were not detected by TUNEL staining.â‘¥Immunocytochemical method showed differentiated NSCs induced by FBS were NeuN,GFAP and O₄ positive respectively.⑦On GRE T₂^*WI only tubes of 1×10⁶ and 1×10⁵ cells presented obvious low signal intensity.On FSE T₂WI only tubes of 1×10⁶ cells presented obvious low signal intensity.On T₁WI there was no obvious difference among all five tubes.Conclusionâ‘ It is feasible,efficient and safe to label NSCs with SPIO-PLL complex.â‘¡NSCs labeled with SPIO-PLL complex can result in a negative enhancement on GRE T₂^*WI and FSE T₂WI,which can be detected by a clinical 1.5 T MR imaging system.The degree of negative enhancement is correlated with the cell number.GRE T₂^*WI is the most sensitive sequence to detect negative enhancement of labeled cells.Experiment four:Magnetic resonance tracking of transplanted NSCs derived from GFP transgenic embryonic mice labeled with SPIO in rat brain after facial nerve injuryObjective To explore the migration of transplanted NSCs derived from GFP transgenic embryonic mice labeled with SPIO in rat brain after facial nerve injury and the feasibility of magnetic resonance tracking of this migration.Methods NSCs were derived from the brain of GFP transgenic embryonic mice and labeled with SPIO.24 adult SD rats were randomly divided into 4 groups:A group, transplanted SPIO labeled NSCs in rat brain after facial nerve injury;B group, transplanted unlabeled NSCs in rat brain after facial nerve injury;C group, transplanted nutrient solution contained SPIO in rat brain after facial nerve injury;D transplanted SPIO labeled NSCs in normal rat brain.1 week following model induction,NSCs stereotaxis transplantation was performed,and the injection point was 11.30 mm to pone of the anterior fontanelle,9.00 mm to dorsal side,at the median line.MR scanning was performed to monitor the transplanted cells after implantation in 3 day and 2,3,4 weeks respectively.After MRI on the fourth week all rats were killed and frozen sections of brain tissues were prepared.Fluorescence microscope and Prussian blue staining of the histological sections were employed to observe the migration of GFP and SPIO co-labeled NSCs and the results were compared with MRI.Resultsâ‘ At grafting region:MRI:there was a round area of low signal intensity in groups A,C and D except B from the third day to the fouth week after transplantation. Prussian blue staining:there were blue staining area in groups A,C and D except B. And this area was matched with the low signal intensity area on MRI;Fluorescence microscope:GFP-positive cells were observed in groups A,B and D except C.In groups A and D GFP-positive cells were matched with blue staining area on Prussian blue staining.â‘¡In the area around facial nucleus of injured side facial nerve(ventral side of right part of pons):MRI:From the third day to the second week after transplantation there was no area of low signal intensity in all groups.From the third week to the fourth week after transplantation there was low signal intensity presented on 3 animals(3/6) of group A.No low signal intensity was observed on the other animals.Prussian blue staining:Only group A had blue staining area which was matched with the low signal intensity area on MRI.Fluorescence microscope:In groups A and B GFP-positive cells were observed migration to this area.And in groups A GFP-positive cells were matched with blue staining area on Prussian blue staining.â‘¢In the area around facial nucleus of healthy side facial nerve(ventral side of left part of pons):MRI:There was no area of low signal intensity in all groups;Prussian blue staining:There was no area of blue staining in all groups;Fluorescence microscope:No GFP-positive cell was observed migration to this area in all groups.â‘£In blood vessel:Some GFP-positive cells were observed in blood vessel near or away from the injection point.⑤The area between grafting region and facial nucleus: No GFP-positive cell or blue staining was observed connecting the two points.Conclusion NSCs co-labeled with SPIO and GFP could migrate into area around nucleus of injured side facial nerve after transplanted into rat brain and 1.5T MRI is feasible to tracking the migration of the labeled neural stem cells.