Study Of Combination Therapy For Rat Model Of AD By Implanting Grafting RhNGF Microspheres And Neural Stem Cells

Alzheimer's disease (AD) is an irreversible, progressive disorder due to brain cells (neurons) deterioration, resulting in cognitive impairment, such as abnormal primarily memory, judgment and reasoning, movement coordination, and pattern recognition. A lot of investigations have showed that the prevalence of AD is strongly associated with aging. As the life span of human beings increases and more people live beyond the age of 65, the number of people with AD is also progressively increased. Contrasting with the enormous toll that this disease puts on patient, his or her caregivers, society as a whole is the lack of an effective therapy at present.One of the neuropathological characteristics of AD is atrophy or loss of cholinergic neuron observed in the basal forebrain (BF). Neurotrophic factors are a good strategy to prevent or reduce the neuronal atrophy or loss. Among the neurotrophic factor family, nerve growth factor (NGF) is the most highly characterized neurotrophic factor for peripheral sympathetic neurons and a subpopulation of sensory neurons. The cholinergic neurons of BF express both the low affinity receptor (P75^NTF) and TrkA receptor, and respond to NGF by increased activity levels of the choline acetyltransferase (CHAT). NGF is widely used for therapeutic studies in the experimental models of AD. However, NGF is a large molecular protein that does not easily cross the blood-brain barrier and has a short biologic half-life. Although infusing NGF solution into the cerebroventricular space via osmotic minipumps showed some therapeutic effects, long-term ICV NGF administration may cause negative side effects. The delivery of NGF to the BF poses a major challenge. New approach for drug formulation is required. One possibility is using controlled release formulations for the targeted delivery of NGF to brain.Neural stem cells have been successfully cultured, which solved the clinical problem of human fetal donors. Neural stem cells can generate neurons, astroglia and oligodendroglia in response to environmental signals, such as neurotrophic factors, retinoic acid and growth factors. The survival and differentiation of neural stem cells are associated with the inducement of the neurotrophic factors in vitro and in vivo. In central nervous system (CNS), the survival, migration and differentiation of neural stem cells is not lack of the microenvironment of the brain. Neural stem cells can differentiate specific neurons of specific areas of the brain under the inducement of the microenvironment.In this study, we treated the rat model of AD by combining rhNGF microspheres with neural stem cells. On the one hand, neural stem cells will differentiate into neurons, which will supplie the degenerative neurons. On the other hand, the rhNGF released from the rhNGF microspheres will promote an/or ameliorate the survival and function of degenerative neurons. Furthermore, the released rhNGF from the microspheres will supply the nourishment for the survival, migration and differentiation of neural stem cells.The following three parts were included in this study:1. The preparation and evaluation of rhNGF microspheres. Microspheres, containing rhNGF and BSA, were prepared by (water-inooil)-in-water (W/O/W) emulsion and solvent evaporation technique with some modification. Briefly, 5 mg protein mixture of rhNGF and BSA (1/2000, w/w) in 100μl distilled water was emulsified in Poly (D, L-lactic-co-glycolic acid) (PLGA) (100mg) solution (3 ml of methylene dichloride and 1 ml of acetone) using sonication for lmin at 30 W over ice-bath. The first w/o emulsion was added to 25 ml of 1% PVA aqueous solution and homogenized at 1000rpm for 5 min over ice-bath. The resulting w/o/w double emulsion was stirred in a hood for 3-4 h to evaporate the organic solution at room temperature. The microspheres were collected by centrifugation, washed three times with distilled water and freeze-dried to obtain a free flowing powder. The rhNGF and FITC-BSA (1/2000, w/w) microspheres were prepared in the same way for in vivo studies. The following variables were designed to assess the effect of technical parameters on particle size, protein loading, and encapsulation efficiency. (1) Microspheres were prepared with a series of protein/PLGA ratios of 5%, 10% and 15% respectively. (2) Microspheres were prepared by adding different additives such as sucrose, PEG, and glycerol in the inner aqueous phase at concentrations of 5 and 10% (w/w) respectively. The results showed rhNGF in PLGA microspheres provided a sustained release formulation with low initial burst (11.4 %) for at least 35 days in vitro. The results showed that the higher the protein/polymer ratio, the higher the protein loading into the microspheres, and the lower the efficiency of protein encapsulation in the microspheres. The encapsulation efficiency could be increased with adding water-soluble additives in the inner aqueous layer prior to the emulsification. The efficiency of protein encapsulation was about 89.7 % without using additive and increased to 97.5 % with the using of PEG. The microencapsulation technique allowed an entrapment of biologically active rhNGF. This is the first report so far of rhNGF-loaded microspheres implanted into BF. The biodegradable rhNGF-loaded microspheres maintained a sustained release of rhNGF for at least 4 weeks in brain tissue.2. The migration and differentiation of neural stem cells in the basal forebrain of the rat model of AD. The single cell suspension from brain tissues of the basal brain of new-born (less than 1 day) rats by gentle mechanical dissociation with the use of trypsin were cultured in DMEM/F12 medium containing 2% B27, EGF and FGF-2 (10 ng/ml). The cell were maintained for 5-7 day at 37℃in 5% CO₂. After 5-7 days, neurospheres were generated and floated in the culture. Unilateral fimbria-fornix (FF) of SD rats was transected to simulate the impairment of cholineric neurons of AD by the lesion of the pathway of septohippocampus. Neural stem cells (25,000 cells/μl×4μl) were injected into rats brain: AP+0.6 ram, LL+0.6 ram, DV-5.5 mm。The results showed neural stem cells could divide and proliferate under the inducement of EGF and FGF-2. After 5-7 d in vitro, large self-renewing and expandable spheres were generated. The proliferating and multipotential differentiation properties of neural stem cells were identified by immunochemistry. BrdU labeling and immunochemistry test were used to confirm the proliferation potential. In vivo studies, after 1 week, most of grafted cells stayed in the needle pathway, which survived well in the host brain, expressing Nestin antigen. After 2 weeks, most of grafted cells migrated neighbouring brain tissue, less of grafted cells expressing Nestin antigen. After 3 or 4 weeks, non-grafted cells express Nestin antigen, most of grafted cells migrated into whole basal forebrain and showed BrdU+NF or BrdU+GFAP.3. The therapeutic effect of combining rhNGF microspherewith neural stem cells on rat AD model. Adult male SD rats (12-20 weeks old, 250-310g) were used in this study. The rats were divided into five groups:â‘ Normal control groups rats (Normal group, n=8);â‘¡FF-lesion groups rats (LES group, n=8);â‘¢RhNGF microspheres group rats (MIC group, n=12);â‘£Neural stem cells group rats (NSC group, n=12);⑤The combination of rhNGF microspheres and neural stem cells group rats (MIC+NSC group, n=12). Neural stem cells (25,000 cells/μl×4μl) were injected into rats brain: AP+0.6mm, LL+0.6mm, DV-5.5mm。Three milligrams of rhNGF microspheres (suspended in 10μl of dispersing medium) were stereotaxically implanted into the BF (coordinates: AP +0.6 mm, LL +0.6 mm, DV-5.5 mm from bregma). The rats survived for four week after FF-lesion, discrimination learning and memory in Y-maze were observed. The morphologic data and percentages of cholinergic neurons of medial septum (MS) and vertical diagonal branch (VDB) were analyzed statistically by SPSS. The results showed after FF lesion, the percentages of cholinergic neurons at the lesion side to the intact side of MS and VDB were significantly lost. In MIC group, the percentages of cholinergic neurons at the lesion side to the intact side of MS and VDB were protected, which were significantly higher than that in the lesion group (p＜0.01). In NSC group, the percentages of cholinergic neurons at the lesion side to the intact side of MS and VDB were protected, which were significantly higher than that in the lesion group (p＜0.01). In MIC+NSC group, the percentages of cholinergic neurons at the lesion side to the intact side of MS and VDB were protected, which were significantly higher than that in MIC or NSC group (p＜0.01). In the behavioral changes, the learning and memory of rats were significantly improved in the MIC+NSC group.Summary: The rhNGF-containing PLGA microspheres were preparaed by a W/O/W emulsion and solvent evaporation technique with some modifications. Using higher protein/polymer ratios in primary emulsions resulted in higher protein contents in the microspheres. The encapsulation efficiency could be increased with adding water-soluble additives in the inner aqueous layer prior to the emulsification. The in vitro rhNGF release lasted for more than 5 weeks. The rhNGF- microspheres maintained a sustained release of rhNGF for at least 4 weeks in brain. Neural stem cells can be obtained from basal forebrain by using free-serum medium adding EGF and FGF-2 (10 ng/ml). After FF lesion the percentages of cholinergic neurons at the lesion side to the intact side of MS and VDB were significantly lost. The loss of cholinergic neurons was associated with the impairment of learning and memory of the rat AD model. The combinative therapy of rhNGF microspheres and neural stem cells could significantly ameliorate, rescue and supply the degenerative neurons of the basal forebrain, and significantly improve the spatial learning of the rat AD model.