1、EXPERIMENTAL STUDIESYutaka Mine, M.D.Department of Neurosurgery,School of Medicine,Keio University, andDepartment of Neurosurgery,Eiju General Hospital,Tokyo, JapanTakuro Hayashi, M.D., Ph.D.Department of Neurosurgery,College of Medicine,University of South Florida,Tampa, FloridaMotoyuki Yamada, M.D
2、.Department of Neurosurgery,Tokyo-Kita Social Insurance Hospital,Tokyo, JapanHideyuki Okano, M.D., Ph.D.Department of Physiology,School of Medicine,Keio University,Tokyo, JapanTakeshi Kawase, M.D., Ph.D.Department of Neurosurgery,School of Medicine,Keio University,Tokyo, JapanReprint requests:Takesh
3、i Kawase, M.D., Ph.D.,Department of Neurosurgery,School of Medicine,Keio University,35 Shinanomachi, Shinjuku-ku,Tokyo, Japan 160-8582.Email: kawasesc.itc.keio.ac.jpReceived, May 27, 2007.Accepted, February 2, 2009.Copyright 2009 by theCongress of Neurological SurgeonsABBREVIATIONS: Ab, antibody; CX
4、CR4, CXC chemokine receptor 4; DA, dopaminergic; DAPI, 4H11541,6-diamidino-2-phenylindole; DARPP-32, dopamine- and 3H11541:5H11541-monophosphate-regulated phosphoprotein 32;DMEM, Dulbeccos modified Eagles medium; ES, embryonic stem; FGF, fibroblast growth factor; GFP,green fluorescent protein; Ig, i
5、mmunoglobulin; M-NESC, mesencephalic neuroepithelial stem cell; M-NP,mesencephalic neural plate; NSC, neural stem cell; OHDA, hydroxydopamine; PD, Parkinsons disease; SN,substantia nigra; TH, tyrosine hydroxylaseENVIRONMENTAL CUE-DEPENDENT DOPAMINERGICNEURONAL DIFFERENTIATION AND FUNCTIONALEFFECT OF
6、 GRAFTED NEUROEPITHELIAL STEM CELLSIN PARKINSONIAN BRAINOBJECTIVE: To physiologically repair damaged neural circuitry using neural trans-plantation, the donor cells must be able to differentiate into the optimal type and num-ber of neurons for the host brain environment. They must also be capable of
7、 functionalregulation by the host brain. These features are important to optimize functional out-come and to minimize side effects. In this study, the differentiation of grafted mesen-cephalic neuroepithelial stem cells in the normal and parkinsonian brain was assessedmorphologically and behaviorall
8、y to confirm the influence of the host brain environ-ment after neural transplantation regarding functional outcome and side effects oftransplants.METHODS: Green fluorescent protein-positive mesencephalic neuroepithelial stemcells were dissected from early rat embryos and transplanted into normal (n
9、 H11005 20)and Parkinsons disease (PD) rat model striata (n H11005 30). The differentiation pattern ofgrafted cells was precisely monitored immunohistochemically, and the functionaleffects of grafted cells on behavior were assessed using an amphetamine-inducedrotation test.RESULTS: The grafted cells
10、 survived in both normal and PD rat striata and differenti-ated into tyrosine hydroxylase-positive cells. In 8-week-old grafts, the number of tyro-sine hydroxylase-positive cells was 3-fold higher in parkinsonian brains than in nor-mal brains. The donor-derived tyrosine hydroxylase-positive cells ex
11、hibited a maturemorphology with long, well-branched cell processes and large cell bodies, especiallyin parkinsonian brains. Also, the process lengths of these cells in parkinsonian brainswere 3.4-fold longer than those in normal brains at 8 weeks after transplantation. Thegrafted PD rats exhibited a
12、 complete recovery from their behavioral defects, and noobvious contralateral rotation to the lesioned and grafted side suggestive of overdose sup-ply from the graft was observed. However, the grafted normal rats did not exhibit anycontralateral rotation to the grafted side suggestive of dopamine un
13、balance.CONCLUSION: Grafted mesencephalic neuroepithelial stem cells can differentiateinto optimal neuron types in response to environmental cues and can affect the behav-ior of PD rats without any side effects.KEY WORDS: Environment, Neural stem cells, Neural transplantation, Neuroepithelium, Regen
14、erative med-icine, Side effectNeurosurgery 65:741753, 2009 DOI: 10.1227/01.NEU.0000351281.45986.76 www.neurosurgery- NEUROSURGERY VOLUME 65 | NUMBER 4 | OCTOBER 2009 | 741Many researchers have reported the possibility of usingneural transplantation for damaged or malfunctioningneural circuitry (4, 2
15、6, 42). Experimentally, some reports,including ours, have indicated that neural transplantations canameliorate behavior in animal models (14, 31, 34), although sideeffects of neural transplantation have been reported in experi-mental studies using Parkinsons disease (PD) rat models (10, 12,24, 54, 5
16、5) as well as in clinical trials using human fetal substan-tia nigra (SN) cells (5, 21).For physiological reconstruction of damaged neural circuitryusing neural transplantation without side effects, the donor cellsmust be able to differentiate into the optimal type and number ofneurons, extend their
17、 axons and form synaptic connections withhost brain tissue, exert functional effects on the host brain, andbe regulated functionally by the host brain (21). These featuresare important for avoiding side effects in neural transplantation,such as dopamine overdose from donor cells in patients withPD.
18、However, estimating the optimal type and number of neu-rons in individual damaged brains can be difficult. Some studiesmentioned above reported side effects during functional exami-nations as a result of dopamine overdose from the transplantedcells in PD rats. Apparently, too many cells may be trans
19、plantedinto the damaged brain.Mesencephalic neuroepithelial stem cells (M-NESCs), a type ofneural stem cell (NSC), compose the mesencephalon in earlyembryos and possess stem cell capabilities, self-renewal, and mul-tipotency (18, 19). M-NESCs can proliferate by fibroblast growthfactor (FGF) 2 admini
20、stration (18), and differentiate neurons,astrocytes, and oligodendrocytes (18, 19). Fresh and FGF2-respon-sive M-NESCs differentiate mostly into neurons and express sev-eral neurochemical markers: glutamate (75.1% H110064.66%79.9% H110061.83%), -aminobutyric acid (9.93% H110061.41%12.4% H110061.94%)
21、, andtyrosine hydroxylase (TH) (1.3% H110060.27%1.41% H110060.69%). Thereare, however, no cholinergic neurons (19). They also possessmany in vivo features required for neural reconstruction, such asgood survival in the host brain (51), migratory capability (16, 50),vigorous neuronal differentiation
22、with the ability to extend longmyelinated axons (19, 36 48, 49, 51), both efferent and afferentanatomic synaptic formation (48), electrophysiologically activeneuronal differentiation, and synaptic formation with postsynap-tic current (48). Thus, we think that of all types of NSCs, M-NESCs are one of
23、 the most promising candidates for neural trans-plantation (19, 48, 51). The influence of the host brainenvironment to grafted NSCs is still uncertain. However, the sideeffects of neural transplantation could be avoided if grafted M-NESCs can differentiate into optimal neurons in response to envi-ro
24、nmental cues and provide functional effects to the patient.In this study, the differentiation of grafted M-NESCs in nor-mal and parkinsonian brain tissue was assessed morphologi-cally and behaviorally to confirm the influence of the host brainenvironment after neural transplantation.MATERIALS AND ME
25、THODSAnimalsSixty-four 4-week-old female Wistar albino rats (Saitama Experi -mental Animal Supply Co., Ltd., Saitama, Japan), weighing 80 to 100 g,were used. Twenty rats were used to assess a normal brain environ-ment and 44 were subjected to 6-hydroxydopamine (OHDA) lesions foruse as PD rats. For h
26、arvesting the stem cells, 8-week-old heterozygousmale transgenic Sprague Dawley rats carrying enhanced green fluores-cent protein (GFP) (green rats) (provided by Masaru Okabe, Ph.D.,Osaka University, under a material transfer agreement via Japan SLC,Inc., Hamamatsu, Japan) (22, 32), weighing 200 to
27、250 g, and wild-type8-week-old female Wistar-Imamichi rats (Saitama Experimental AnimalSupply Co., Ltd.) (46, 47), weighing 150 to 200 g, were used. All exper-imental animals were handled according to the guidelines of theAnimal Experimental Committee of Keio University.Preparation of Host AnimalsFo
28、ur-week-old female Wistar albino rats were used to create hemi-parkinsonian model rats using a previously described method (30, 41,53). In total, 44 rats were anesthetized with pentobarbital sodium (30mg/kg intraperitoneally; Dainippon Sumitomo Pharma Co., Ltd.,Osaka, Japan) and treated with atropin
29、e sulfate (0.01 mg/kg intraperi-toneally; Mitsubishi Tanabe Pharma Corp., Osaka, Japan). Each animalwas placed in a stereotaxic apparatus (Narishige Scientific InstrumentLab., Tokyo, Japan). 6-OHDA (Sigma Chemical Co., St. Louis, MO)solution (16 g free base in 8 L of 0.05% ascorbate saline; Daiichi
30、PureChemicals Co., Ltd., Tokyo, Japan) was injected unilaterally into theright SN according to the following stereotaxic coordinates frombregma: anterior, H110024.1 mm; lateral, 1.4 mm; and ventral, 7.2 mm belowthe surface of the dura mater. The nose bar was set 3.3 mm below theinteraural line. The
31、animals methamphetamine-induced rotationbehavior was examined at 3 weeks after injection (see below, underBehavioral Assessment). As a result, 36 6-OHDAlesioned rats satisfiedthe criterion and were used as PD rats, and 8 rats were excluded.Another 20 normal Wistar albino rats (normal rats) were used
32、 as recip-ients of M-NESCs to compare the morphology and function of thegrafted M-NESCs in PD rats.Harvesting of Stem CellsM-NESCs were obtained from embryos as described below. Eight-week-old male green rats were mated overnight with 8-week-oldfemale Wistar-Imamichi rats in estrus, which can be def
33、initely con-firmed in this particular strain (46, 47). The morning after mating wasdesignated as embryonic day 0.5 (E0.5). Both heterozygous and wild-type E10.5 embryos were taken by cesarean section from deeply anes-thetized Wistar-Imamichi rats. The purpose of this mating usingWistar-Imamichi rats
34、 was to confirm the exact embryonic day, and toobtain green-fluoresced embryos, i.e., GFP-positive M-NESCs, to iden-tify the donor-derived cells in the host brain after transplantation. Theembryos were examined under a fluorescent microscope, and onlygreen-fluoresced embryos were selected. M-NESCs w
35、ere harvestedfrom the mesencephalic neural plate (M-NP) of E10.5 rats as previ-ously reported (14, 48, 49, 51). Briefly, embryos were immediately pre-treated with 0.5% trypsin (Invitrogen Corp., Carlsbad, CA) and 0.25%pancreatin (Invitrogen Corp.) for 15 minutes at 4H11034C after delivery tofacilita
36、te complete removal of mesenchyme from head primordia. Afterwashing in phosphate-buffered saline, the head primordia were trans-ferred to 0.7 mg/mL trypsin inhibitor (Boehringer-Mannheim,Mannheim, Germany). The M-NP was dissected out and isolated byremoving the rostral prosencephalon, caudal mesence
37、phalon, and lat-eral mesenchyme in serum-free Hanks buffer (Invitrogen Corp.). Thedissected tissue was membranous and consisted of homogeneous M-NESCs that were positive for anti-nestin antibody and anti-FGF recep-tor antibody (18). The dissected M-NPs were dissociated by manualpipetting and centrif
38、uged at 800 rpm for 5 minutes. One pregnantMINE ET AL.742 | VOLUME 65 | NUMBER 4 | OCTOBER 2009 www.neurosurgery-Wister-Imamichi rat usually had 6 to 8 green-fluoresced embryos, andwe obtained the same number of M-NPs. One M-NP obtained approx-imately 7 H11003 103M-NESCs, thus we obtained approximat
39、ely 4.2 to 5.6H11003 104M-NESCs from 1 pregnant rat. The cells were resuspended inDulbeccos modified Eagles medium (DMEM) (Invitrogen Corp.) atthe cell density of 2 H11003 107cells/mL for transplantation.TransplantationImmediately after harvesting, 10-L aliquots of cell suspension(including 2 H11003
40、 104M-NESCs) described above were stereotacticallytransplanted per rat per group using a Hamilton syringe to slowlyinject cells into the right striatum (ipsilateral to the 6-OHDA lesion) of8-week-old PD rats (n H11005 30) (PD rats with M-NESCs) or normal Wistaralbino rats (n H11005 20) (normal rats
41、with M-NESCs) at 4 or 5 weeks afterthe production of 6-OHDA lesions. The transplantation site was 0 mmanterior to the bregma, 3 mm lateral to midline, and 5 mm below thesurface of the brain (48, 49). As a control, the remaining 8-week-old PDrats (n H11005 6) were injected with 10 L of serum-free DME
42、M (InvitrogenCorp.) in the same manner (PD rats with DMEM). No immunosuppres-sant was used during the experiments in this study.Behavioral AssessmentThree weeks after injection of 6-OHDA, the effectiveness of dopa -minergic (DA) innervation was evaluated on 3 successive days by theappearance of meth
43、amphetamine-induced (3.0 mg/kg body weight,intraperitoneally; Dainippon Sumitomo Pharma Co., Ltd.) ipsilateralabnormal rotation behavior. The criterion for abnormal rotation wasmore than 10 full turns per minute over 30 minutes on each examinationday. The 6-OHDAlesioned rats that satisfied this crit
44、erion were selectedas PD rats in this study. At 2, 4, 6, 8, 10, and 12 weeks after transplanta-tion, the efficacy of transplantation was assessed in all rats by anmethamphetamine-induced rotation test. The rotational behavior of nor-mal rats with M-NESCs was also examined by the same manner ifredund
45、ant dopamine supply from the graft caused contralateral rotationto the grafted side.ImmunohistochemistryFrom the study population of 64 rats, a total of 42 rats were killed at2, 4, or 8 weeks after transplantation. Twenty-six were PD rats with M-NESCs and 16 were normal rats with M-NESCs. The remain
46、ing rats (4PD rats with M-NESCs; 4 normal rats with M-NESCs; 6 PD rats withDMEM) were not killed in order to assess the methamphetamine-induced rotation behavior. The selected rats were overdosed with pento-barbiturate (Dainippon Sumitomo Pharma Co., Ltd.) and perfusedthrough the heart with physiolo
47、gical saline and then 4% paraformalde-hyde in phosphate-buffered saline. The brains were removed, kept in 4%paraformaldehyde overnight at 4H11034C for postfixation, and then cryopro-tected in 10% sucrose in phosphate-buffered saline followed by solu-tions of 15%, 20%, and 30% sucrose until they sank
48、. Then, they wereembedded in Tissue-Tek OCT compound (Sakura Finetechnical Co., Ltd.,Tokyo, Japan) and frozen in liquid nitrogen and dry ice. Subsequently,the brains were cryosectioned coronally using a cryostat (Microtomecryostat HM505 E; Microm International GmbH, Walldorf, Germany)into 20-m-thick
49、 slices and mounted on slides coated with poly-L-lysine.These sections were double stained immunohistochemically using thefluorescent staining method as described elsewhere (48, 49, 51). Primaryantibodies (Ab) were polyclonal rabbit anti-GFP Ab (1:500; Medical Sigma) or mouse anti-dopamine-and 3H11541:5H11541-monophosphate-regulated phosphoprotein 32 (DARPP-32, stri-atal neuronal marker) Ab (1:500; generous gift from Prof. P. Greengard,Rockefeller University, NY) or mouse antimicrotubule-associated protein2 Ab (1:100; Upstate, Temecula, CA) or mouse
