| BackgroundTourette syndrome (TS) is a neurobehavioural disorder occurring in children and is characterized by multiple motor and vocal tics. Tics are stereotypic, involuntary, purposeless and repetitive movements which persist for years in TS and wax and wane in frequency and intensity during their natural courses. Although TS is a benign neuropsychiatric disorder, nearly half of the cohort can not be free of tics even until 18 years of age. In most cases, tics are self-limited or can be treated by behavioural or pharmacological therapy. However, for some individuals, tics can cause lifelong impairment and about 5% of TS patients have life-threatening symptoms, which were defined malignant TS. These symptoms are intractable to conservative treatment and various attempts have been made to treat these patients through neurosurgical procedures. Recently, there has been increasing interest in deep brain stimulation (DBS) as a potential treatment for patients with severe, refractory tics. Nonetheless, the use of DBS for the treatment of TS is limited because of complications related to the surgical procedure and the infrequent hardware. Therefore, new therapy should be explored for those unfortunate patients who are significantly impaired.During recent years, stem cell-based therapy has been proved promising as a potential treatment for many neurological disorders. Neural stem cells (NSCs) are considered a heterogeneous population of mitotically active, self-renewing, multipotent and immature progenitor cells. Animal experiments suggest that if NSCs are injected into the brain or even the bloodstream, the transplanted cells would survive and migrate to damaged portion of the nervous system to incorporate into working neural circuits, and the tested animals display significant functional improvement. Therefore, NSC replacement is thought to be a promising alternative therapeutic approach for treating neurological disorders. It may breakthrough some of the existing limitations of traditional pharmaceutical approaches. Most important of all, neural stem cell therapy is a good choice for the treatment of neural diseases whose exact pathogenesis are unclear.The definitive pathophysiological mechanism of tics, at molecular and cellular level, is still unknown. However, structural and functional neuroimaging, neurophysiological, and post-mortem studies have shown the dysfunction of the basal ganglia and related cortico-striato-thalamo-cortical circuits, and the dopaminergic neuronal system. Taking these scholarship into consideration, we hypothesized that intrastriatal tansplantation of NSCs could replace disfunctioned or damaged cells in the central nervous system of TS, reduce the frequency and intensity of stereotypic behaviors and further help regain homeostasis.ObjectivesTo investigate the methods of isolation, cultivation, purification, expanding, BrdU marking and identification of neural stem cells from striatum of fetal Wistar rat and observe the proliferation and differentiation of neural stem cells. Study the effect of neural stem cell transplantation on Wistar rats which were microinfused with the sera of TS patients. Observe the growth and differentiation of transplanted cells in the rat brain and the therapeutic effects of neural stem cells on the stereotypic behavior of TS rat.MethodsNSCs were isolated from rat embryonic brain (E13). Embryonic striatum tissue was microdissected under a stereo microscope and minced into small pieces, and then mechanically triturated through a 26-gauge needle and dissociated to single cell suspension. Cells were cultured in serum-free basic medium DMEM: F12 supplemented with human recombinant epidermal growth factor (EGF) (20 ng/ml), fibroblast growth factor-basic (bFGF) (20 ng/ml), B27 (2%), penicillin-streptomycin 1% . Cells were incubated with 5% CO2 at 37℃. Within 2-3 days the cells grew as free floating neurospheres and half of the growth medium was replaced. Expanded neurospheres were collected by centrifugation and dissociated into single cells once every 5-6 days with 0.25% trypsin and mechanical trituration. NSCs were co-incubated with 5-bromodeoxyuridine (BrdU, 10μg/ml) for 24h prior to transplantation. Then BrdU-labeled NSCs (passage number 3-4) were harvested and enzymatically dissociated into single cells. Cells with higher than 85% viability were centrifuged and resuspended in basic medium supplemented with B27 at a density of 5×10~5 cells/μl and then stored on ice until grafting. To determine the phenotype of cultured cells, the cells were separately immuno-stained with anti-Nestin antibody, anti-MAP2 antibody or anti-GFAP antibody.Blood samples were drawn from patients with TS and were sent for further enzyme-linked immunosorbent assays (ELISA) to a laboratory. The selection of sera for infusion was based on ELISA optical density readings against caudate homogenate, that is, we selected the highest serum OD readings for TS subjects and the lowest OD readings for the control infusion subgroup. A total of thirty-two Wistar rats were used in the study and randomly divided into four groups: sham (microinfused with normal sera), TS alone (microinfused with TS sera), TS plus PBS vehicle injection (TS+PBS) and TS plus NSCs grating (TS+NSCs) (n=32, i.e., n=8 for each group).Rats were deeply anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic apparatus with the incisor bar set at 3.5mm below the interaural line. Then using aseptic surgical technique, the skull was exposed and holes were drilled where appropriate and 28 gauge guide cannulae were implanted into bilateral striatum. Coordinates for cannulae placements were anterior-posterior 2.0mm from bregma and medial-lateral 4.0mm and dorsoventral -7.0mm from the skull. One week later, osmotic mini pump filled with PBS was connected to each cannula by a polyethylene tube loaded with 50μl of undiluted TS or control serum under sterile conditions. Sera were microinfused at a rate of 0.5μl /hour for 72 hours. The neural stem cell suspension (2μl) was injected bilaterally using a 5μl Hamilton syringe with a 26-gauge needle at the sera-infusion site after the pumps were removed. Rat movements were video and audio taped at the end of the rat's 12-hour light cycle for 30 min. Several categories of stereotypy including bites (teeth touching the cage, wood chips, vacuous chewing or other objects except the body), taffy pulling (raises of the forepaw to the mouth and face), self-gnawing, licking not associated with grooming, grooming, head shaking, paw shaking, rearing and episodic utterances (EU) were recorded. Stereotypic movements were recorded at 1 day, 7 days, 14 days and 21 days after transplantation.Three weeks after transplantation, rats were given a lethal dose of chloride hydrate (600 mg/kg) and transcardially perfused with cold 0.9% NS followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4, 4℃). Brains were removed from the cranium and post-fixed in PFA prior to sectioning. Rat brain sections were embedded in paraffin and 4-μm coronal sections were prepared. All the immunostaining processes were finished according to the instruction of histostain?-DS kit. Primary antibodies which included mouse monoclonal to GFAP—astrocyte marker, mouse monoclonal to MAP2—neuron marker and mouse monoclonal to Nestin—neural stem cell marker were added.ResultsFollowing the isolation, a close-to-single-cell suspension was obtained, which consisted of viable small, round cells. In suspension cultures containing EGF and bFGF, rat striatal NSCs began to proliferate and adherent clonal clusters of cells (10 - 20 cells/clone) started to appear after 48 - 72 h. The clusters divided rapidly and generated large colonies, i. e. neurospheres. These floating neurospheres displayed a spherical shape, and the cells forming the neurosphere were phase bright. These spheres were successfully passaged by dissociation and reculture. This procedure resulted in a rapid increase in cell number. The neural cells dissociated from E13 rat striatum had the capacity to proliferate and form neurospheres. The neurosphere could express Nestin, gernerate MAP2 or GFAP positive neural cells.Eight children were selected from 20 patients with TS and their sera were used for rat striatal microinfusion. Similarly, eight specimens of sera were selected from 20 control samples. The selection of sera for infusion was based on ELISA optical density readings against caudate homogenate, that is, we selected the highest serum OD readings for TS subjects and the lowest OD readings for the control infusion subgroup. The mean±SD optical density readings in serum of 8 TS subjects selected for microinfusion was 0.904±0.177 and 0.252±0.193 for the 8 selected controls.Serologic studies of children with TS have detected the existence of anti-neural antibodies, which can induce striatal dysfunction. In our study, stereotypies were successfully induced in rats by intrastriatal microinfusion of TS sera under noninflammatory conditions. After infusion of TS sera, stereotypic behaviors in rats increased significantly. Marked differences were observed in stereotypies of TS rats compared to control rats after microinfusion. TS rats exhibited significant increases in bites, taffy-pulling, gnawing, licking, head shaking, paw shaking, rearing and episodic utterance. All rats in four groups were observed at 1 day, 7 days, 14 days, and 21 days after their transplantations. Stereotypic behaviors of each rat were recorded and counted through each 30 minutes of observation period. The statistical analysis suggested that TS rats had increased stereotypic behaviors compared to normal rats (Sham). Using a repeated measurements analysis of variance (ANOVA), we found that the overall model had significant group (F=354.13, p<0.0001) and day (F= 106.01, p<0.0001) effects, as well as a (group×day) interaction (F=21.38, p<0.0001), indicating varying degrees of differences among the groups and across days. A model with just TS+NSCs and TS+PBS groups indicated significant differences between groups(F=36.63, p=0.0001) and a strong day effect(F=100.23, p<0.0001). Importantly, animals with NSCs grafts showed a significant decrease in stereotypic behaviors at 14 and 21 days. Tukey-Kramer post hoc test showed significant differences between TS+NSCs and TS+PBS groups beginning at 2 weeks post-transplantation. However, rats receiving NSC grafts (TS+NSCs) still had higher stereotypic behavior counts than sham controls (p<0.0001), which indicated that the animals' impairment did not fully recover in a short period of 3 weeks.Histological analyses of recipient rat brains transplanted with NSCs or injected with PBS (vehicle) were given 3 weeks after transplantation. In order to trace the transplanted cells, we used BrdU labeling, so that the presence of the implanted NSCs within striatum of TS rats identifiable for the nuclei of the grafted cells were BrdU-positive and stained with purple. In the TS+NSCs rats, BrdU-positive cells were prevalent in the striatum sections near the injection site, suggesting the survival of the cells. Some of the grafted cells expressed both BrdU and MAP-2 and others expressed both BrdU and GFAP. These results suggested that NSCs used as grafts grew at the injection site and differentiated into neurons or astrocytes. Our results also indicated that a considerable portion of the transplanted cells had differentiated to mature neurons in vivo, which may lead to histological and functional reconstitution in the CNS. Besides, few BrdU and Nestin double positive cells were found, indicating undiferentiation of a small number of the grafted cells.ConclusionsNeural stem cells were isolated from fetal rat striatum and were certificated as a heterogeneous population of mitotically active, self-renewing, multipotent, immature progenitor cells. These cells expressed the intermediate filament protein Nestin and grew as neurospheres in vitro. Neural stem cells had the capacity to differentiate into neurons and gilocytes..In our study, stereotypies were successfully induced in rats by intrastriatal microinfusion of TS sera under noninflammatory conditions. Our results showed that intrastriatum transplanted rat NSCs relieved stereotypic behaviors. These results suggested the potential of using NSCs intrastriatum as a clinical treatment for TS. Neural stem cells survived in the brain of TS rat and part of them differentiated into neurons and gliocytes. These newly generated neurons from the transplanted NSCs might have played a role for the neural networks in the damaged areas. No signs of immunologic rejection or non-neural tissue growth were found in the host rat brain after transplantation.
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