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Neuroprotective effects of bone marrow stromal cells on rat organotypic hippocampal slice culture model of cerebral ischemia

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Neuroprotective effects of bone marrow stromal cells on rat organotypic hippocampal slice culture model of cerebral ischemia
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  Neuroprotective effects of bone marrow stromal cells on rat organotypichippocampal slice culture model of cerebral ischemia Chi Zhong a , Zhen Qin a, *, Chun-Jiu Zhong b , Yang Wang c , Xin-Ya Shen c a  Institute of Neurology, Huashan Hospital, Fudan University, Shanghai 200040, China b  Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai 200032, China c  Department of Anatomy, Histology and Embryology, Shanghai Medical College, Fudan University, Shanghai 200032, China Received 3 December 2002; received in revised form 17 February 2003; accepted 19 February 2003 Abstract Organotypic hippocampal slice cultures prepared from newborn rats were maintained in vitro for 9 days. Cultures were then exposed to 30min of combined oxygen–glucose deprivation (OGD). After OGD, the area covered by neurites was decreased. The dead cells of hippocampal slices in the ischemia group were 40.4% at day 3 and 41.6% at day 7 after OGD. The ultrastructure of the CA1 region of theslices was seriously damaged. While hippocampal slices were cultured in combination with bone marrow stromal cells (MSCs), the averagearea covered by neurites was comparatively increased. The dead cells were only 25.2% at day 3 and 27.1% at day 7 after coculture. Thedamage of the ultrastructure of the CA1 region in the coculture group was reduced significantly. Thus, in an in vitro model of simulatedischemia, MSCs can promote the outgrowth of neurites from hippocampal slices and alleviate cell damage. The neuroprotective effect mightbe mediated through diffusible neurotrophic factors secreted from MSCs. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords:  Bone marrow stromal cells; Hippocampus; Organotypic culture; Ischemia; Coculture Bone marrow stromal cells (MSCs) have the capacity of self-renewal and multiple potentiality of differentiation[12]. MSCs delivered to ischemic brains of animals werereported to be therapeutically beneficial in recent studies [4,9,10,15]. In the present study, the ischemic organotypichippocampal slices placed onto Millipore inserts (0.4  m Mpore size) were cultured in combination with MSCs todetermine whether MSCs can protect hippocampal slicesfrom ischemic injury when they are not in direct contactwith each other.Primary cultures of MSCs were obtained from the femursand tibias of adult Sprague–Dawley rats. The rats wereanesthetized with ketamine. The bones were dissected cleanof attached muscles and the marrow was expelled from themarrow cavity using D-Hank’s balanced salt solution(BSS). Bone marrow was pooled and the cells were pelletedby gentle centrifugation. Then, 2  £  10 6 nucleated marrowcells were seeded into each 25 cm 2 tissue culture flask inDulbecco’s modified Eagle’s medium (DMEM) sup-plemented with 20% fetal bovine serum, 2 mM glutamine,100 units/ml penicillin and 100 mg/ml streptomycin. Thestromal cells were isolated by their adherence to plastic.MSCs were grown at 37  8 C, 100% humidity in 5% CO 2 . Themedium was changed every 3–4 days. After the cells hadgrown to confluence (about 10 days), they were passagedfour to six times by being detached with 0.25% trypsin and 1mM EDTA for 5 min and replated after dilution to about1:2. Three days before coculture with hippocampal slices,the MSCs were harvested and replated in each well of thesix-well culture trays at 5000 cells/cm 2 .Organotypic hippocampal slice cultures were preparedfrom 1-day-old Sprague–Dawley rat pups based on themethod of Stoppini et al. [13] with several modifications.The animals were deeply anesthetized by hypothermia, andtheir brains were removed. The hippocampi were bluntlydissected out and cut into transverse slices (400  m M thick).The slices were then transferred onto 24 mm diametermembrane inserts (Transwell-clear, Corning Coster Cor-poration, USA) and put into six-well culture trays with 1.5ml slice culture medium per well. The culture trays were 0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved.doi:10.1016/S0304-3940(03)00255-6Neuroscience Letters 342 (2003) 93–96www.elsevier.com/locate/neulet* Corresponding author. Tel.:  þ 86-21-62489999/6542; fax:  þ 86-21-62483421. E-mail address:  georgezqin@hotmail.com (Z. Qin).  stored at 37  8 C in a 5% CO 2  enriched atmosphere, 100%humidity incubator. The slice culture medium consists of 50% DMEM, 25% heat-inactivated horse serum (GibcoBRL Life Technol Inc., USA) and 25% Tyrode’s BSSsupplemented with 4.5 mg/ml glucose and 1 mM glutamine.The medium was changed every 3 or 4 days and oxygen–glucose deprivation (OGD) was performed after 9 days invitro.In vitro ischemia was simulated by anoxia combinedwith glucose-free medium. The hippocampal slices werefirst washed three times with Hank’s balanced salt solution(HBSS). Then, HBSS was replaced with deoxygenated,glucose-free DMEM (Gibco BRL Life Technol Inc., USA).After that, the cultures were placed into an air-tight chamberthrough which 95% N 2  /5% CO 2  gas was passed at 5–10l/min. The temperature of the chamber was kept at 37  8 C.After 10 min of gas flow, the chamber was sealed and placedinto a 37  8 C incubator for 30 min. Reperfusion wassimulated by replacing the exposure medium with normalmedium and placing it back into the incubator undernormoxic conditions. The slices subjected to ischemia werecultured for 1, 3 and 7 days respectively after OGD. For thecoculture group, the ischemic hippocampal slices on themembrane insert were placed over the MSCs in the well.They were cultured in combination for 1, 3 and 7 daysseparately. For estimation of neurite outgrowth of thehippocampi, slices were examined under phase-contrastmicroscopy. Images were captured for analysis using theLeica image analysis system. The area of the substratecovered by neurites was measured. For each time point, 12cultures were examined. As shown in Fig. 1, the averagearea covered by neurites was decreased when hippocampalslices were cultured alone. Meanwhile, the neurites grewslowly and sparsely. But when ischemic hippocampal sliceswere cultured in combination with MSCs, the area coveredby neurites was increased gradually. Three to 7 days aftercoculture, the average area of the substrate covered byneurites was significantly increased. The results indicatethat MSCs might release some factors which are able toenhance the growth of neurites after ischemia.On the third day of reperfusion, transmission electronmicroscopy of six pieces of the hippocampal slices wasperformed for every group. Most neurons in the CA1 regionof the ischemia group showed peripheral chromatincondensation, disruption of organella structure and cellmembrane with shrinking of cytoblast. Most neurites weredestroyed (Fig. 2A). In coculture group, fewer neurons weredead. The karyotheca was intact and the chromatin wasevenly distributed in most neurons. The structure of organellae looked normal on the whole and the neuritescould be found easily (Fig. 2B). This suggests that MSCsmay play a key role in protecting the hippocampal slicesfrom ischemic damage on neurons through releasing sometrophic factors.The fluorescent exclusion dye propidium iodide (PI) wasused to study the neuronal death [11]. PI is excluded fromhealthy cells but enters dead cells following their loss of membrane integrity. It binds to exposed DNA and becomeshighly fluorescent. The slice culture medium containing 5 m g/ml PI was added tothe wells of culture plates. PI stainingwas checked 20 min later. The excitation light wavelengthwas 510–560 nm, and the emission light wavelength was590 nm. The area of the hippocampal slice was measuredfrom a transmission image on which it was clearly visible.The area of the hippocampal slice in which PI fluorescencewas detectable above background was determined using theLeica image analysis system. The percentage area of eachhippocampal slice in which PI fluorescence occurred wascalculated at 1, 3 and 7 days after ischemia. The extent of dead cells varied with the region and duration of reperfu-sion. On the first day of reperfusion, dead cells were foundin all regions, but predominantly in the CA1 region,followed by CA2, CA3 and dentate gyrus. A total of 23%of the cells were dead on the first day of reperfusion, 40.4%on the third day and 41.6% on the seventh day (Fig. 3). Butwhen ischemic hippocampal slices were cultured incombination with MSCs, there was only a small numberof dead cells in the CA1 region, and a few dead cellsscattered in CA2, CA3 and dentate gyrus. The total amountsof dead cells were only 17.6% on the first day of coculture,25.2% on the third day and 27.1% on the seventh day (Fig.3). This suggests that MSCs might protect the ischemichippocampal slice cultures when they are cultured incombination.Organotypic hippocampal slice culture is an in vitromodel adopted to investigate ischemia related neuronaldamage. The cultures retain much of the synaptic connec-tivity and extracellular microenvironment that exists in vivo[3]. Our present observations indicate that MSCs haveneuroprotective effects on ischemic hippocampal sliceswhen they are cultured in combination immediately afterischemic insult. The protective effect appeared on the firstday of coculture and became more significant on the third Fig. 1. Effects of MSCs on neurite outgrowth from hippocampal slices asmeasured by quantitative image-capture analysis. Ischemic hippocampalslices were cultured alone or cocultured with MSCs for 1, 3 and 7 daysseparately. The average area covered by neurites was decreased whenhippocampal slices were cultured alone. MSCs promoted the neuriteoutgrowth obviously 3–7 days after coculture. The average area of thesubstrate covered by hippocampal neurites was significantly increased.Data are expressed as means ^ SEM. **Significant compared to theischemia group;  P  ,  0 : 001, one-way ANOVA with Tukey post-hoc test. C. Zhong et al. / Neuroscience Letters 342 (2003) 93–96  94  and seventh days. During coculture, MSCs in the wells wereseparated from hippocampal slices on the membrane inserts.Therefore, the protective effect was probably exerted bysome diffusible factors. The interaction between MSCs andthe hippocampal slice cultures may increase the productionof trophic factors from MSCs and attenuate the injury to theischemic hippocampal slices. MSCs express the neural cell-adhesion molecule neuropilin and neurotrophic factors(NTFs) like nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), macrophage colony-stimulat-ing factor (M-CSF), granulocyte-macrophage colony-sti-mulating factor (GM-CSF), stem cell factor (SCF),interleukin-6 (IL-6), transforming growth factor  b  (TGF- b ), bone morphogenetic protein-1 (BMP-1), fibroblastgrowth factor (FGF), etc. Growing a series of murine bonemarrow stromal cell lines in the presence of cytokines andgrowth factors (TNF- a , TNF- b , IL-1 a , FGF-1, FGF-2,FGF-4, EGF, IL-4, IL-6 and TGF- b ) changes the expressionlevels of cytokines in MSCs. The most striking effect wasproduced by TNF- a  and IL-1 a  which increased theexpression of M-CSF, IL-6, and TGF- b  in MSCs signifi-cantly [7]. So, inflammatory cytokines such as TNF- a  andIL-1 a produced by ischemic neuronal cells [2] can stimulatesecretion of NTFs from MSCs while ischemic hippocampalslices are cultured in combination with MSCs. In addition,under pathologic conditions such as ischemia, NTFs, e.g.BDNF, basic fibroblast growth factor (bFGF), glial-derivedneurotrophic factor (GDNF), NGF, and TGF- b 1, are alsoinduced in brain cells [1]. These NTFs also can promoteMSCs to secrete NGF, BDNF, M-CSF, etc. When humanMSCs (hMSCs) were cocultured with traumatic brain injury(TBI) extracts of rat brain in vitro, the production of BDNF,NGF, vascular endothelial growth factor (VEGF) andhepatocyte growth factor (HGF) was increased in TBI-conditioned hMSCs cultures in a time-dependent mannerindicating a responsive production of these growth factorsby the hMSCs [5]. hMSCs also express fibronectin andcollagen type-I when they are grafted into the cortexsurrounding the area of infarction 1 week after cortical brainischemia in rats [15]. It is known that NTFs are involved innormal maintenance and survival of neuronal cells afterdifferentiation. Constant secretion of NTFs keeps cellsurvival signals activated and death signals inactivated,resulting in cell protection [1]. However, it needs to beinvestigated further which NTFs plays the most importantrole in the neuroprotection and if the protection is anadditive effect of several NTFs. Fig. 2. Electron photomicrographs of the CA1 region in hippocampal slices of the ischemia group (A) and the coculture group (B). Scale bar, 1  m m.Fig. 3. Effects of MSCs on cell death in hippocampal slices. Neuronal deathis expressed as % cell damage as defined by PI fluorescence imaging.Ischemic hippocampal slices were cultured alone or cocultured with MSCsfor 1, 3 and 7 days separately. Total dead cells increased more significantlyat 3 and 7 days after ischemia. The neuroprotective effect was significantwhen MSCs were cocultured with ischemic hippocampal slices. Data areexpressed as means ^ SEM. **Significant compared to the coculturegroup;  P  ,  0 : 001, one-way ANOVA with Tukey post-hoc test. C. Zhong et al. / Neuroscience Letters 342 (2003) 93–96   95  NGF is able to stimulate neurite outgrowth from thehippocampal explants [6]. PC12-E2 cells, a stable variantsubclone from native cell populations, produce neurites in arapid and transcription-independent manner upon exposureto NGF or bFGF. A similar morphological response of PC12-E2 cells to IL-6 was also reported [14]. BDNF induces neurite outgrowth from SY5Y neuroblastoma cellsin a dose-dependent manner as well [8]. Our results indicatethat MSCs significantly promote the neurite outgrowth fromischemic hippocampal slices which suggests that MSCsmight contribute to neuronal sprouting and synaptogenesisof hippocampal slices. This may be one of the structuralbases for reorganization of deficient neuronal function. Theneuroprotection might be mediated through diffusiblefactors secreted from MSCs. References [1] K. Abe, Therapeutic potential of neurotrophic factors and neural stemcells against ischemic brain injury, J. Cereb. Blood Flow Metab. 20(2000) 1393–1408.[2] F.C. Barone, G.Z. Feuerstein, Inflammatory mediators and stroke:new opportunities fornovel therapeutics, J. Cereb.Blood FlowMetab.19 (1999) 819–834.[3] P.A. Buchs, L. Stoppini, D. Muller, Structural modificationsassociated with synaptic development in area CA1 of rat hippocampalorganotypic cultures, Dev. Brain Res. 71 (1993) 81–91.[4] J. Chen, Y. Li, L. Wang, Z. Zhang, D. Lu, M. Lu, M. Chopp,Therapeutic benefit of intravenous administration of bone marrowstromal cells after cerebral ischemia in rats, Stroke 32 (2001)1005–1011.[5] X.Chen,M.Katakowski,Y.Li,D.Lu, L.Wang,L. Zhang,J.Chen,Y.Xu, S. Gautam, A. Mahmood, M. Chopp, Human bone marrowstromal cell cultures conditioned by traumatic brain tissue extracts:growth factor production, J. Neurosci. Res. 69 (2002) 687–691.[6] H.J. Clarris, V. Nurcombe, D.H. Small, K. Beyreuther, C.L. Masters,Secretion of nerve growth factor from septum stimulates neuriteoutgrowth and release of the amyloid protein precursor of Alzhei-mer’s disease from hippocampal explants, J. Neurosci. Res. 38 (1994)248–258.[7] S.P. Dormady, O. Bashayan, R. Dougherty, X.M. Zhang, R. Basch,Immortalized multipotential mesenchymal cells and the hematopoie-tic microenvironment, J. Hematother. Stem Cell Res. 10 (2001)125–140.[8] R.H. Fryer, D.R. Kaplan, L.F. Kromer, Truncated trkB receptors onnonneuronal cells inhibit BDNF-induced neurite outgrowth in vitro,Exp. Neurol. 148 (1997) 616–627.[9] Y. Li, J. Chen, L. Wang, M. Lu, M. Chopp, Treatment of stroke in ratwith intracarotid administration of marrow stromal cells, Neurology56 (2001) 1666–1672.[10] Y. Li, M. Chopp, J. Chen, L. Wang, S.C. Gautam, Y.X. Xu, Z. Zhang,Intrastriatal transplantation of bone marrow nonhematopoietic cellsimproves functional recovery after stroke in adult mice, J. Cereb.Blood Flow Metab. 20 (2000) 1311–1319.[11] D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate andnon-glutamate receptor mediated toxicity caused by oxygen andglucose deprivation in organotypic hippocampal cultures, J. Neurosci.15 (1995) 7702–7711.[12] D.J. Prockop, Marrow stromal cells as stem cells for nonhematopoie-tic tissues, Science 276 (1997) 71–74.[13] L. Stoppini, P.A. Buchs, D. Muller, A simple method for organotypiccultures of nervous tissue, J. Neurosci. Methods 37 (1991) 173–182.[14] Y.Y. Wu, R.A. Bradshaw, Induction of neurite outgrowth byinterleukin-6 is accompanied by activation of Stat3 signaling pathwayin a variant PC12 cell (E2) line, J. Biol. Chem. 271 (1996)13023–13032.[15] L.R. Zhao, W.M. Duan, M. Reyes, C.D. Keene, C.M. Verfaillie, W.C.Low, Human bone marrow stem cells exhibit neural phenotypes andameliorate neurological deficits after grafting into the ischemic brainof rats, Exp. Neurol. 174 (2002) 11–20. C. Zhong et al. / Neuroscience Letters 342 (2003) 93–96  96
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