The Immunohistochemical Characterization Of Human Fetal Olfactory Bulb And Olfactory Ensheathin Cells In Culture

ObjectiveThe therapeutic olfactory ensheathing cells (OECs) transplantation in clinical studies has been used to different diseases in central nervous system (CNS). In fact, the OEC transplantation in clinic is a mixture of olfactory bulb cells;they even have not demonstrated that they have such a subpopulation yet. The purpose of this study is to do more in-depth research and discussion of clinical transplantation of OECs used in the content of the various components and the distribution of OECs in the olfactory nerve layer(ONL) and Glomerular Layer of Human Fetal OB.Methods1. Immunohistology:six olfactory bulbs from human fetuses in the 20th gestation week were fixed in freshly prepared 4% paraformaldehyde for 2 hr. Following fixation,specimens were cryoprotected into 20% sucrose solution for 24 hr and frozen.To determine the characterization of OECs in human fetal OB, the bulbs were cut into coronal in the rostralcaudal axis.8μcoronal sections were cut with a cryostat, mounted the sections on glass slides (Fisher). To stain the sections,the primary antibodies used in P75/S100β/Laminin/Nestin or GAFP. Secondary antibodies were goat-anti-mouse Alexa 488 or 546,goat-antirabbit Alexa 488 or 546,goatanti-mouse Cy5.To stain nuclei,we used Hoechst 33342.2. Cultures of human fetal olfactory ensheathing cells:For cell culture, another six of olfactory bulbs from human fetuses in the 20th gestation week were isolated, representing cells left over from transplantation procedures. The bulbs were placed in ice-cold Hank's Balanced Salt Solution (HBSS). After washing the bulbs three times with HBSS,the bulbs were diced into small fragments and digested in trypsin-EDTA (0.05% w/v) at 37℃or 10 min. An equal volume of DMEM-F12 with 10% certified fetal bovine serum (D/F-10S) was added to stop the digestion. The cells were cultured on cover slips with D/F-10S media for 7 days before fixation and staining.3.Immunostaining to identify OECs in culture:Cell cultures were fixed with 4% paraformaldehyde for 10 min and then washed with PBS.After incubating the cells in the blocking solution, the primary antibody was added for 1 hr and secondary antibody for 30 min. The nuclei were stained with Hoechst 33342.4.Photomicrography and cell counting:The sections and the culture dishes were imaged with a Zeiss LSM510 confocal microscope. Large images were scanned in high resolution and stitched together using Zeiss Axio Vision software. The cell counting in cell culture was performed by recognized different colors marked cells on the photomicrographs on the screen of Zeiss LSM510 confocal microscope, using Zeiss Axio Vision.Result1.The distribution of OECs in the olfactory nerve layer of human fetal OB: The entire ONL expressed P75NTR positive immunostaining, the outer layer expressed intensely and the inner layer expressed dimly. Double labeling with different cell markers showed P75NTR appeared in the outer part of OBL layer. GFAP was intensely expressed in both inner and outer layers, but not co-expression.2. The distribution of OECs in the glomerular layer of human fetal OB:The glomerular layer was located in the deep of ONL.The P75NTR-positive OECs were clearly present around the glomeruli. The intensity for GAP-43 was homogeneous around the glomeruli. MAP-2 marked neuronal dendrites in the center of glomeruli, especially in the medial-anterior region of the olfactory bulb.3.Immunohistochemic characteristics of human fetal OB cells in culture: The human fetal OB cell culture was separated into two groups.One was the small pieces of OB tissue culture, another was OB cells culture. The P75NTR-positive and about 46.04% of S100b-positive OB-cells;Nearly all of the GFAP-positive OB-cells;P75NTR-positive OB-OECs were co-immunolabel-ed with NCAMs; some of the P75NTR-positive OB-OECs as well as other OB cells were coimmunolabeled with nestin; no P75NTR-positive OB-OECs was coimmunolabeled with FBN-1.ConclusionOur data indicated that P75NTR-positive OECs present in the outer nerve layer of OB,and participate in glomerular formation in fetal human (20th week) olfactory bulb,entering the glomerular layer with ingrowing olfactory axons, encircling and sending processes into developing glomeruli.Our study further confirmed the classification and percentage of OB cell cultures that were predominantly immunopositive for P75NTR, GFAP, and S100b in human fetus OB cell culture at mid-gestation.P75NTR,GFAP,and S100b immunohistoch-emic staining are still necessary to identify OB-OECs in cell culture before CNS restoration. ObjectiveIn our study we observe the onset, disease process, survival time and the behavioral improvement for experimental ALS rats after OECs were tansplantated and explore the mechanism of neuroprotection and remyelination. It will provide the experiment basis for clinical application of this new strategy.Methods1.Identification of mutant SOD1G93A transgenic ratsSprague-Dawley pups were genotyped 3 weeks of age. A small piece of tail was cut, the DNA was abstracted, and then PCR was performed by using 4μL of this solution. Positive and negative controls were used in every PCR reaction.2.Preparation of OECsThe bulbs were placed in ice-cold Hank's Balanced Salt Solution (HBSS). After washing the bulbs three times with HBSS, the bulbs were diced into small fragments and digested in trypsin-EDTA (0.05% w/v) at 37℃or 10 min. An equal volume of DMEM-F12 with 10% certified fetal bovine serum (D/F-10S) was added to stop the digestion.The cells were cultured on cover slips with D/F-10S media at 37℃for CO2.3.OEC transplantationRats were anesthetized with 4% (0.12ml/kg) chloral hydrate.Laminectomy was performed between vertebrae T6 and T8.Then 5μL (1×105) OECs in DF12 medium were injected into the dorsal funiculus of thoracic spinal cord (T7) through a glass micropipette at depths of 0.7 mm and 0.4 mm(0.5μL).4.Clinical assessment and Inclined board testDisease onset,survival time, body weight and inclined board test were measured throughout the study, at 100 days.5.Preparation of tissue sections for confocal microscopy and motor neuron countAll SOD1G93A rats were histologically evaluated at 2 weeks (age= 114 days) and 4 weeks (age=128 days,1 day after disease onset)after the date of treatment. All SOD1G3A rats in each group as well as the Wild-type controls were anesthetized with 4% chloral hydrate and perfused transcardially with 4% paraformaldehyde in PB. The motor neuron count in the ventral gray matter was performed at 4 weeks after OEC transplantation (age=128 days,1 day after disease onset).6.ImmunohistochemistryThe ChAT and NF immunohistochemistry were performed to identify and quantify the expression of the active of motoneurons and axon. The tissues for immunohistochemistry were obtained at 4 weeks after OEC transplantation. The sections were incubated overnight at 4℃with rabbit anti-ChAT or rabbit anti-NF200. The section was treated with second antibody conjugated with fluorescent dye in the dark for 1 h at room temperature. Sections were coverslipped with 30% glycerin and observed under confocal microscope. Digitalized microphotographs of immuno-fluorescent sections were saved.7.Western blotFor western blot, rats were anesthetized and killed with an overdose of pentobarbital, perfused with ice-cold PBS from the left ventricle. The spinal cord was removed, frozen and stored at-80℃until protein extraction. Tissues were homogenized in ice-cold lysis buffer. The protein content was determined by Bio-Rad protein assay. Equal amounts of protein then were loaded.Proteins were then transferred to nitrocellulose and the membrane was blocked. The nitrocellulose was then incubated with different antibodies overnight at 4℃. The membrane was treated with horseradish peroxidase-conjugated secondary antibody, and then exposed to X-ray film. The X-ray films were scanned and the optical density was determined by Bio-Rad Image analysis.Results1.The identification of OEC before and after transplantationTwo week after transplantation,OECs showed the diffuse distribution of GFP-expressing OECs in the spinal cord of SOD1 G93A rats. Four weeks after transplantation, a farther dispersion (8.4mm) of GFP-expressing OECs indicated the cell migration rather than pressure-induced spreading.2.Clinical assessment and behavioral testAnimals were tested on a Rotarod apparatus weekly as a measure of onset of disease. There was no significant difference between OECs+SOD1G93A rats and SOD1G93A rats (or Medium+SOD1G93A rats) in terms of their stability on the Rotarod at 105 days.Kaplan Meier survival analysis revealed a significant increase in survival of OEC+SOD1G93A rats versus SOD1G93A rats or Medium+SOD1G93A rats. A significant difference was found between OEC+SOD1G93A and SOD1G93A (or Medium+SOD1G93A) at 7-10 days (age=134-137 days) after disease onset (p>0.05) in body weights and inclined test.3.Motor neurons quantificationNissl staining and motor neuron count in per ventral horn of thoracic spinal cord showed a significant motor neuron loss in SOD1G93A rats and in Medium +SOD1G93A rats when compared with wild type rats(p<0.001). SOD1G93A rats which received OEC transplantation showed much less neuronal loss and collapse than control rats. The ChAT amount of immunohistochemic staining was same as that of Nissl staining.4..Western blotIt was shown after quantification, the protein levels of ChAT in both SOD1G93A and Medium+SOD1G93A were significantly decreased (versus wild type rats, p<0.05), but the ChAT protein level in OECs transplantation rats significantly increased than that in both SOD1 G93A and Medium+SOD1G93A (p<0.05).5.Evidence that transplanted OECs ccan remyelinate CNS axons in the spinal cord of SOD1G93A rats.The double staining for GFP (transplanted OECs) and Neurofilament (neuronal fibers) indicated that the transplanted GFP expressing cells and produced peripheral-like myelin (green fluorescence marked) which were near and far away from the injecting site.ConclusionOECs can prolong the survival and improve functions for transgenic SOD1G93A rats. It reveals that the transplanted OECs not only can migrate at least 8.4mm away from the injecting site, but also can protect motor neurons from death, remyelin the regenerated never fibers.