Author contributions: T.R.D.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; T.A.E.: collection and/or assembly of data, provision of study material or patients, and final approval of manuscript; L.T. and F.N.: provision of study material or patients and final approval of manuscript; J.H., A.Z., A.E., and A.-K.L.: collection and/or assembly of data and final approval of manuscript; B.G. and G.P.D.: manuscript writing and final approval of manuscript; J.W.: conception and design, financial support, and final approval of manuscript; D.M.H.: administrative support, manuscript writing, and final approval of manuscript; M.B.: financial support, administrative support, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS March 30, 2012.
Novel therapeutic concepts against cerebral ischemia focus on cell-based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell-based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein-transduced with TAT-heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT-Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT-Hsp70-transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT-Hsp70-transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood-brain-barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long-term neuroprotection. STEM CELLS2012;30:1297–1310
Neurogenesis persists in the adult rodent brain within the subventricular zone (SVZ) [1–3]. Cerebral ischemia stimulates neurogenesis [4–6], and newly formed neurons release cellular mediators into the extracellular space that promote neurological recovery [7, 8]. Unfortunately, survival and differentiation rates of new-born cells are low [4–6, 9, 10]. Therefore, exogenous NPC transplantation strategies have been used to overcome limitations of endogenous neurogenesis [7, 11, 12].
Transplantation of NPCs ameliorates ischemic brain injury in rodents [7, 8, 13–17], but its therapeutic benefit is hampered by low survival rates of grafted cells [18, 19]. Since cell transplantation is usually done after stroke , grafted cells are not exposed to ischemic insults directly. Rather, cells are transplanted into a nonfavorable tissue environment, in which they are exposed to oxidative stress and inflammation [21, 22], which may cause protein misfolding and cell death [23, 24].
Regarding clinical translation, the most appropriate route of NPC transplantation is an important but unresolved question. Among different transplantation routes, intravenous cell delivery has been used due to its simplicity and clinical practicability [8, 25, 26], although achieving low intracerebral cell numbers [15, 27]. Therefore, local intracerebral injections are widely used, which can enhance the number of transplanted intracerebral cells but lack clinical utility due to invasive delivery procedures [28–32]. Until recently, only two studies systematically compared the effect of different NPC transplantation routes on postischemic outcome. However, these studies are limited to a maximal observation period of 1 week and lack an analysis of underlying cellular mechanisms [33, 34].
To elucidate consequences of NPC delivery strategies, we herein submitted mice to cerebral ischemia and transplanted NPCs 6 hours later either intravenously (systemically) or intrastriatally (intracerebrally), followed by an analysis of brain injury and remodeling for up to 8 weeks. In order to promote survival of grafted cells, NPCs were protein-transduced with the chaperone Hsp70, which reduces apoptosis and inflammation after hypoxic-ischemic injury [23, 24]. Since cellular entry of Hsp70 is poor, transduction was achieved in vitro using the membrane-permeable and neuroprotective TAT-Hsp70 fusion protein before transplantation [10, 35]. This study has explicit meaning for future cell-based stroke therapies, since it highlights the importance of cell transplantation routes, implying different mechanisms by which transplanted NPCs induce poststroke neuroprotection.
MATERIALS AND METHODS
Animals and Experimental Groups
Experimental procedures were in accordance with the National European Institutes of Health guidelines for the care and use of laboratory animals and approved by local authorities. For all experiments, male C57BL/6 mice (11–13 weeks, 23–27 g; Charles River, Sulzfeld, Germany (www.criver.com) were used. The total number of animals was 437, which were assigned to eight treatment paradigms (Table 1). Treatment was performed in a blinded manner. A schematic display for intracerebral transplantations and the in vivo experimental paradigm is given in Supporting Information (Supporting Information Fig. S1). Survival rates were 100% for all animals surviving 1, 4, 14, or 28 days for each experimental condition, and the amount of animals used per experimental group as described in Materials and Methods. The survival rate of animals receiving systemic or intracerebral injections of phosphate buffered saline (PBS) was 78.6% (11/14) on day 56 for both groups. Survival rate after intracerebral transplantation of native NPCs, TAT-HA-transduced, or TAT-Hsp70-transduced NPCs was 84.6% (11/13) for each group. For animals that had received systemic delivery of TAT-Hsp70-transduced NPCs survival was 91.6% (11/12), whereas for the remaining experimental groups with a 56 days survival, the survival rate was 100%. Assessment of cell proliferation was performed on animals surviving either 28 days or 56 days. These mice received 11 consecutive intraperitoneal (i.p.) injections of 5-bromo-2-desoxyuridine (BrdU; 50 mg/kg b.wt.) on days 8–18.
Table 1. groups and numbers of animals used for statistical analysis
Animals were assigned to four treatment groups for both systemic and intrastriatal delivery of adult NPCs or PBS, respectively. All injections were performed 6 hours after stroke onset. Five survival periods were defined for each group. Time points given refer to induction of stroke, which was assigned as day 0. The number of animals given reflects the amount of surviving mice used for final analysis of experimental results.
TAT-Hsp70 and TAT-hemagglutinin (TAT-HA), which is also included in the TAT-Hsp70 construct serving as a negative control, were prepared under native conditions as described . Briefly, the recombinant genes were expressed in Escherichia coli strain BL21 (DE3) pLysS (Novagen, Madison, WI, www.novagen.com) and proteins were isolated in 10 mM Tris, pH 10, 20% glycerol, 274 mM NaCl, 0.1% Pluronic, and 0.02% Tween 80. Bacterial debris was removed by centrifugation and the cell extracts were purified by affinity chromatography using Ni-tris-carboxymethyl-ethylene-diamine . Protein was eluted by stepwise addition of binding buffer containing increasing concentrations of imidazole. The column eluate was purified from imidazole by gel filtration (Sephadex G-25 M, GE Healthcare Bio-Sciences AB, Munich, Germany, www.gehealthcare.com). According to this procedure, TAT-Hsp70 is highly stable after cell transduction as no protein degradation could be detected 24 hours after protein application, suggesting that the intracellular half-life time is at least a few days . For transplantation of NPCs, in vitro incubation period with either TAT-Hsp70 (250 nM) or TAT-HA (250 nM) was 4 hours.
Preparation and Cultivation of Adult SVZ-Derived NPCs
NPCs were isolated from the SVZ of 19 6–8 weeks old male nontransgenic C57BL/6 mice for in vitro experiments . For in vivo experiments, 346 transgenic green fluorescence protein positive (GFP+) animals (C57BL/6-Tg ACTB-enhanced green fluorescence protein (EGFP), 1Osb/J; JAX Laboratory, Bar Harbor, Maine, www.jax.org; male, 6–8 weeks old) were used. GFP expression was under control of the actin promoter, which allows reliable and stable tracking of transplanted NPCs. The SVZ was microdissected under stereomicroscopic control (Zeiss, Jena, Germany, www.zeiss.de), minced into small pieces, and then mechanically triturated and dissociated into a single-cell suspension. Cells were cultured in serum-free basic Dulbecco's modified Eagle's medium (DMEM)/F12 (PAA, Linz, Austria, www.paa.com) supplemented with epidermal growth factor (EGF, 2 μg/ml), basic fibroblast growth factor (bFGF, 2 μg/ml), and penicillin-streptomycin (Invitrogen, Frankfurt, Germany, www.invitrogen.com). Cells were incubated with 5% CO2 at 37°C. The growth factors were supplemented every 2–3 days and cells were passaged via accutase (Invitrogen) digestion for 30 minutes at 37°C with a resuspension every 10 minutes. Thereafter, cells were centrifuged and resuspended in conditioned media. Neurosphere passages were done every 7–10 days and cells used for transplantation were derived from passage 4.
Quantitative Analyses of NPCs and Neurosphere Differentiation Assay
For estimation of neurosphere numbers, 10,000 primary NPCs (P0) and cells from passage 4 (P4) were plated in six-well plates and cultured for 7 days with/without TAT-HA (250 nM) or TAT-Hsp70 (250 nM). Thereafter, neurosphere numbers and neurosphere diameters were quantified. Cells were then treated with accutase in order to achieve single-cell suspensions followed by determination of total cell numbers. For differentiation analysis, NPCs from P0 and P4 that had been cultured for 7 days (native or transduced with 250 nM TAT-HA or 250 nM TAT-Hsp70) were gently triturated and plated onto 24-well plates (40 cells per microliter) containing coverslips coated with 1 mg/ml poly(D-lysine) (Sigma-Aldrich, Taufkirchen, Germany, www.sigmaaldrich.com) according to modified protocols [38, 39]. Differentiation medium consisted of DMEM/F12 supplemented with 2% B27 (Invitrogen), 1% fetal bovine serum, and penicillin-streptomycin. For Western blot analysis, cells were lysed (50 mM Tris at pH 8.0, 150 mM NaCl, and Triton 1%) on day 7 postplating. Lysates were centrifuged and supernatants were used for SDS-PAGE. Equal amounts of protein (75 μg) were diluted in 6× sample buffer, boiled and loaded onto 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes, which were immersed in blocking solution (5% dry milk powder in TBS-T [0.1% Tween 20 + Tris-buffered saline]; 1 hour at room temperature [RT]). The following primary antibodies were used: polyclonal rabbit antinestin (1:2,000), polyclonal rabbit antiglial fibrillary acidic protein (GFAP; 1:10,000), polyclonal rabbit anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; 1 μg/ml), and antibeta tubulin III (1 μg/ml; all obtained from Abcam, Cambridge, UK, www.abcam.com). Membranes were incubated with a peroxidase-coupled, goat anti-rabbit secondary antibody (1:2,000; Abcam), washed several times, immersed in enhanced chemiluminescence solution, and exposed to enhanced chemiluminescence-Hyperfilm (Amersham Biosciences, Freiburg, Germany, www.amershambiosciences.com).
Oxygen-Glucose-Deprivation of Cultured NPCs
Experimental procedures were performed as previously described . NPCs were passaged and 100,000 cells were first preincubated for 4 hours with conditioned cell culture medium containing TAT-Hsp70 (250 nM). Then, the medium was substituted by a glucose-free crystalloid solution (“Thomajodin” plus 1 mM mannitol; Deltapharm, Dortmund, Germany) containing TAT-Hsp70 (250 nM). Cells were incubated in a hypoxic chamber (1% O2; remainder 5% CO2 and 94% N2) for 45 minutes and reincubated in normal glucose and TAT-Hsp70 (250 nM) containing cell culture medium for 24 hours. Cell viability was assessed using a LIVE/DEAD-Viability/Cytotoxicity-Assay kit (Lonza, Basel, Switzerland, www.lonza.com). TAT-HA (250 nM; negative control) and PBS served as controls for TAT-Hsp70. Controls were kept under normal cell conditions without oxygen-glucose-deprivation (OGD) for the duration of the experiment.
Induction of Transient Focal Cerebral Ischemia
Cerebral ischemia was induced using middle cerebral artery (MCA) occlusion . Animals were anesthetized (0.8%–1.5% isofluran, 30% O2, and remainder N2O), and rectal temperature was maintained at 36.5°C–37.0°C using a feedback-controlled heating system under continuous control of blood flow changes by means of a laser Doppler flow (LDF) system (Perimed, Jarfalla, Sweden, www.perimed-instruments.com). Occlusion of the left MCA was achieved using a 7-0 silicon-coated nylon monofilament (180 μm tip diameter; Doccol, Redlands, CA, www.doccol.com), which was withdrawn after 45 minutes to induce transient ischemia. LDF recordings continued for additional 15 minutes to monitor appropriate reperfusion.
Transplantation of NPCs
Mice received either intravenous (systemic) or intrastriatal (intracerebral) injections of NPCs or PBS 6 hours after induction of stroke. Systemic injection of NPCs (106 cells in 100 μl PBS) or PBS (100 μl) was performed via cannulation of the right femoral vein. For intracerebral stereotactic injections of PBS (5 μl) or NPCs (5 × 105 cells in 5 μl PBS), surgeries were performed in mice that were anesthetized with ketamine (10 mg/kg) and xylazine (25 mg/kg). Thereafter, animals were placed in a stereotactic apparatus (Kopf Instruments, Tujunga, CA, www.kopfinstruments.com) and fixed accordingly. The skull was exposed, and a hole was drilled at the appropriate position on the ischemic left hemisphere. Injections (0.4 mm anterior, 1.8 mm lateral and 3.5 mm ventral from bregma) were performed using 10 μl Hamilton syringes (Bonaduz, Switzerland, www.hamiltoncompany.com) with a rate of 1 μl/minute. The syringe was kept in place for additional 5 minutes after injection before removal.
Analysis of Poststroke Brain Injury and Immunohistochemistry
Infarct volumes were analyzed on day 4 (n = 5 per group), for which brains were removed and cut into slices of 2 mm each. Slices were stained with 2,3,5-triphenyltetrazolium chloride (2%), and a computer-based analysis of infarct volumes was done using the freely available software ImageJ (NIH; http://rsbweb.nih.gov/ij) by subtracting the area of the nonlesioned ipsilateral hemisphere from that of the contralateral side. Infarct volume sizes were calculated by integration of the lesioned areas. Postischemic brain edema was measured as the increase of ipsilateral hemispheric volume in comparison to the contralateral hemisphere.
For immunohistochemical analysis, animals were i.p. injected with chloralhydrate (420 mg/kg b.wt.) and transcardially perfused with 4% paraformaldehyde at days 4 (n = 5–6), 14 (n = 7), 28 (n = 8–9), and 56 (n = 7). The brains were removed, shock-frozen in liquid nitrogen, and 16-μm thick coronal cryostat sections were prepared. Quantitative analyses for immunohistochemical stainings were performed defining regions of interest (ROIs) within the ischemic basal ganglia. Stereotactic coordinates were 0.14 mm anterior, 2.5–3.25 mm ventral, and 1.5–2.25 mm lateral from bregma. Three sections per animal and ROI were used. For quantitative analysis of proliferating BrdU+ cells, sections were exposed to blocking solution and subsequently stained with a monoclonal mouse anti-BrdU antibody (1:400; Roche, Mannheim, Germany, www.roche.de) or a monoclonal rat anti-BrdU antibody (1:400; Abcam). Since quenching of fluorescence signal of GFP+ NPCs occurs during section processing, a polyclonal rabbit anti-GFP antibody (1:2,500; Abcam) was used to enhance GFP signal intensity. For differentiation analysis of GFP+ or BrdU+ cells, double staining was performed against BrdU/GFP and a polyclonal goat antidoublecortin antibody (1:50; Santa Cruz Biotechnology, Heidelberg, Germany, www.scbt.com), a polyclonal rat anti-GFAP antibody (1:500; Zymed, Germany), a monoclonal mouse anti-CNPase antibody (1:400; Millipore, Abingdon, UK, www.millipore.com), a monoclonal mouse anti-NeuN antibody (1:1,000; Millipore), or a monoclonal mouse antinestin antibody (GFP counterstaining only; 1:500; Millipore). In order to exclude engulfment of GFP+-transplanted NPCs by microglia, double staining against GFP and IB4 was done on day 4 using a rat biotin-conjugated anti-IB4 antibody (1:100; Vector, Peterborough, UK, www.vectorlabs.com). All antibodies were incubated for 18 hours at 4°C. Thereafter, the sections were incubated for 1 hour at RT. For double staining with BrdU, the following secondary antibodies were used: goat anti-mouse Cy-3 (1:400; Dianova, Hamburg, Germany, www.dianova.com) or goat anti-rat Alexa 594 (1:400; Dianova) for BrdU staining, goat anti-rat Alexa 488 (1:250; Invitrogen, Germany) or donkey anti-goat Alexa 488 (1:250; Invitrogen) for GFAP or doublecortin (Dcx) staining, goat anti-mouse Alexa 488 (1:100; Jackson ImmunoResearch, Newmarket, UK, www.jireurope.com) for CNPase staining, and goat anti-mouse Alexa 488 (1:400; Invitrogen) for NeuN staining. For double staining with GFP, the secondary antibodies were as follows: goat anti-mouse Cy-3 (1:100; Jackson ImmunoResearch) for NeuN, CNPase and nestin staining as well as goat anti-rat Cy-3 antibody (1:200, Abcam) for GFAP staining. Double staining against Dcx was done using a donkey anti-goat Cy-3 secondary antibody (1:500; Dianova). Photos for differentiation analysis with subsequent three-dimensional reconstruction were made using a Zeiss microscope equipped with an Apotome and the corresponding AxioVision software.
For assessment of brain injury on day 4, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was done. The staining was performed incubating the sections with proteinase K (7 minutes at 37°C) followed by exposure to the TdT enzyme reaction according to the manufacturer's manual (Roche). Sections were stained with a streptavidin-Alexa-488-conjugated secondary antibody (2 hours at RT; Invitrogen) and analyzed. In order to assess viability of BrdU+ cells, double staining using the rat anti-BrdU antibody and the TUNEL-protocol was performed. Further analysis of brain injury on days 4, 28, and 56 was performed by determination of neuronal density, that is, after quantitative analysis of NeuN+ cells within defined ROIs as stated above. Cell numbers for each specific staining were recalculated and are given as total amount of cells per square millimeter.
Assessment of glial scar surrounding the ischemic territory was measured in four defined ROIs after GFAP-staining using a modified protocol as previously described . Images were obtained from the indicated ROIs, and the gliotic area was measured for each ROI using Image J software. Data are given as mean area (mm2) out of four ROIs.
Assessment of Poststroke Functional Recovery
Motor coordination deficits were analyzed using the rota rod, tight rope, and the corner turn test. All behavioral tests were performed on the same animals, which were also used for immunohistochemical analysis on day 56. Behavioral tests were performed in a blinded manner. One day before induction of stroke, animals were trained before the beginning of the actual tests on day 7, 14, 28, and 56. Both rota rod and tight rope test were performed as previously described . Using the rota rod test, animals were put on an accelerating treadmill (TSE Systems, Bad Homburg, Germany, www.tse-systems.com; 3-cm diameter) with an accelerating speed of 4–40 rpm. The maximum speed was achieved after 260 seconds, and maximum testing time was 300 seconds. The time until animals dropped was registered and statistically analyzed. For the tight rope test, animals were placed on a 60-cm long rope grasping the string with their forepaws. Maximum test time was 60 seconds, and results were scored from 0 (minimum) to 20 (maximum) according to a validated score , depending on the time animals spent on the rope and whether or not they reached the platform. Rota rod and tight rope tests were performed twice at each time point and means were calculated. For the corner turn test, two vertical boards were attached at one side with an angle of 30°, and each mouse was tested for the side chosen over 10 trials per test day. Whereas healthy animals leave the corner without side preference, mice suffering from stroke preferentially turn to the left, nonimpaired body side [41, 42]. The laterality index was calculated according to the following formula: (number of left turns − number of right turns)/10.
Zymography of Matrix Metalloproteases
Left ischemic hemispheres (n = 4 per condition; 24 hours poststroke) were homogenized in cold lysis buffer (basic buffer) containing 50 mmol/l Tris-HCl (pH 7.6), 150 mmol/l NaCl, 5 mmol/l CaCl2, 0.05% BRIJ-35, 0.02% NaN3, and 1% Triton X-100. Homogenates were centrifuged at 4°C at 12,000g for 5 minutes and supernatants were incubated with a 1:10 volume of gelatine-Sepharose 4B for 1 hour at 4°C. After centrifugation, pellets were resuspended in elution buffer (basic buffer containing 10% dimethyl sulfoxide and 20% volume of lysis buffer); purified samples were analyzed by zymography. Protein concentrations were determined by the bicinchoninic acid method (BCA kit, Thermo Scientific, Karlsruhe, Germany, www.thermoscientific.com). Separation of matrix metalloprotease (MMP)-2 and MMP-9 as pro-form and active form was performed using Novex Zymogram Gels (Invitrogen) according to the manufacturer's instructions. Samples were incubated in nonreducing sample buffer (0.4 mol/l Tris, pH 6.8, 5% SDS, 20% glycerol, and 0.05% bromphenol blue) for 10 minutes at RT and then loaded onto 10% SDS polyacrylamide electrophoresis gels containing 0.1% gelatin. After electrophoresis, samples were incubated with 2.5% Triton X-100 twice for 20 minutes, equilibrated with developing buffer (Invitrogen), and incubated for 18 hours at 37°C. Gels were stained with Coomassie Blue for 30 minutes and destained in washing solution (30% methanol and 10% acetic acid). White bands on a dark background indicated zones of digestion corresponding to the presence of pro-MMPs and activated MMPs on the basis of their molecular weight. As standards, 0.1 ng of human pro-MMP-9 and human pro-MMP-2 (Merck Biosciences, Darmstadt, Germany, www.merckgroup.com) and 0.01 ng of activated MMP-9 and activated MMP-2 (Merck Biosciences) were used. Gels were scanned and densitometrically analyzed.
Analysis of Blood-Brain-Barrier Permeability
Mice (n = 6 per condition) received intravenous bolus injections of 2% Evans blue dye (2 ml/kg b.wt.) via tail vein cannulation 22 hours poststroke . Two hours later, animals were sacrificed by transcardiac perfusion with PBS. Left hemispheres from nonischemic animals that had received no treatment served as reference for extravasal Evans blue dye contents. Brains were removed and separated into hemispheres. Left (ischemic) hemispheres were weighed, homogenized in 2 ml of 50% trichloroacetic acid, and centrifuged at 10,000 rpm for 20 minutes. The extracted Evans blue dye was further diluted with ethanol, and the fluorescence signal was measured with a luminescence spectrophotometer (exc. = 620 nm, em. = 680 nm). An external standard (62.5–500 ng/ml) was used for calculation of Evans blue dye contents, which is given as (μg) Evans blue dye per (g) tissue.
Determination of Thiobarbituric Acid-Reactive Substances
Oxidative stress was assessed 24 hours poststroke in brain homogenates from left ischemic hemispheres (n = 4 per condition), which were obtained using lysis buffer as described above. Formation of reactive oxygen species (ROS) leads to peroxidation of fatty acids of phospholipids contained within the cell membrane. During peroxidation, thiobarbituric acid (TBA) reactive substances (TBARS) are generated such as malondialdehyde (MDA). MDA reacts with TBA resulting in a chromogenic compound whose absorption is photometrically measured at λ = 532 nm . Samples were incubated with cold 80% trichloroacetate solution (volume ratio of 5:1) and centrifuged at 7,500g for 5 minutes. Supernatants were incubated with 1% TBA solution (pH 7.0; volume ratio of 2:1) at 95°C for 10 minutes followed by additional centrifugation at 5,000g and subsequent photometric analysis. The extent of TBARS formation is expressed as MDA equivalents using 1,1,3,3-tetramethoxypropan as standard.
ELISA for Measurement of Growth Factors
For ELISA experiments (n = 4), samples were obtained on days 4 and 56 as described for measurement of TBARS. Levels of growth factors such as vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, www.rndsystems.com), nerve growth factor (Promega, Mannheim, Germany, www.promega.com), brain-derived neurotrophic factor (BDNF; Promega), glial cell line-derived neurotrophic factor (GDNF; Promega), bFGF (R&D Systems), and EGF (R&D Systems, Minneapolis, MN, www.rndsystems.com) were measured using commercial mouse ELISA kits according to the manufacturer's instructions.
All data are given as mean ± SD. For comparison between two groups, the Student's t test was used, whereas for comparison between multiple groups, a one-way analysis of variance followed by the Tukey's post hoc test was performed. A p value of <.05 was considered to be statistically significant.
TAT-Hsp70 Does Not Affect NPC Proliferation and Differentiation but Protects from Hypoxic-Hypoglycemic Cell Injury
Protein delivery using TAT technology is highly efficient and has been successfully tested on various cell types, with often 100% transduction rates in cultivated cells . Previously, treatment of neuroblastoma cells with TAT-Hsp70 (250 nM) achieved at least fourfold higher intracellular levels of TAT-Hsp70 compared to endogenous Hsp70. In this study, we again detected TAT-Hsp70 in cellular lysates after treatment with recombinant protein (Supporting Information Materials and Methods; Supporting Information Fig. S2). Thereafter, effects of both the recombinant protein and cell passaging on NPC proliferation, differentiation, and susceptibility to OGD were analyzed in vitro before cell transplantation.
Neurosphere formation rates were independent of both TAT-Hsp70 and cell passaging (Fig. 1A–1C). Likewise, neurosphere diameters did not significantly differ between treatment groups. As for P0, mean neurosphere diameters were 89.9 ± 27.2 (native NPCs), 107.3 ± 34.1 (NPCs + TAT-HA), and 98.5 ± 24.7 nm (NPCs + TAT-Hsp70). In P4, mean diameters were 93.6 ± 19.4 (native NPCs), 96.9 ± 27.8 (NPCs + TAT-HA), and 101.3 ± 29.1 nm (NPCs + TAT-Hsp70). Furthermore, total cell numbers did not significantly differ between nontreated and transduced NPCs from both passages (Fig. 1D).
Assessment of NPC differentiation (Fig. 1E, 1F) revealed high protein abundance of the neural stem/progenitor cell marker nestin and the astroglial marker GFAP, whereas protein abundance of the neuronal marker β-tubulin III and the oligodendroglial marker CNPase was low. However, no difference between the experimental groups was observed, that is, neither cell passaging nor TAT transduction affected cell differentiation.
TAT-Hsp70-mediated neuroprotection was analyzed in NPCs from P0 and P4 that were exposed to a 45-minute OGD with subsequent recultivation under standard cell culture conditions (Fig. 1G). Whereas TAT-Hsp70-transduced NPCs from passage P0/P4 showed little cell injury, less than 30% of native and TAT-HA-transduced NPCs survived OGD injury.
TAT-Hsp70 Enhances Numbers of Transplanted NPCs After Intracerebral Injection Without Affecting Cell Differentiation
Native or transduced GFP+ NPCs were transplanted systemically or intracerebrally 6 hours after stroke (Fig. 2). Cells were treated ex vivo before transplantation with either TAT-HA or TAT-Hsp70. Intracerebral transplantation within the ischemic striatum resulted in high numbers of GFP+ NPCs under each condition (Fig. 2A). Although the number of grafted NPCs gradually declined over time in all experimental groups, animals that had received TAT-Hsp70-transduced NPCs always showed significantly more transplanted cells (Fig. 2A). The latter typically formed agglomerations at the site of transplantation without significant migration (Fig. 2C–2I). On the contrary, systemic transplantation of NPCs (native/transduced) yielded lower numbers of NPCs under each experimental condition as compared to intracerebral cell delivery (Fig. 2B). Likewise, the number of transplanted cells gradually decreased over time. Transduction of NPCs with TAT-Hsp70, however, did not significantly increase the number of transplanted cells after systemic injection within the ischemic hemisphere.
Differentiation states of transplanted NPCs (Supporting Information Fig. S3) were not affected by TAT-Hsp70 on days 4 and 56 (Fig. 2J, 2K). No colocalization between GFP and the mature neuronal marker NeuN was observed. Noteworthy, counterstaining between GFP and the microglial (and also monocyte) marker IB4  revealed very low rates of colocalizations on day 4 after stroke (Fig. 2J), suggesting that transplanted NPCs are not significantly engulfed by activated microglia.
NPC-Mediated Poststroke Neuroprotection
Infarct volume analysis revealed that both intracerebral and systemic transplantation of NPCs yielded significant neuroprotection on day 4 poststroke, which was independent of NPC transduction states (Fig. 3A–3C). Likewise, edema formation was significantly decreased in animals that had received either intracerebral or systemic transplantation of native or transduced NPCs (Fig. 3D). In line with this, analysis of neuronal density and TUNEL+ cells on day 4 yielded increased neuronal density and reduced numbers of TUNEL+ cells after either intracerebral or systemic injection of native/transduced NPCs (Fig. 3E, 3F). Assessment of long-term neuroprotection after intracerebral transplantation, however, revealed that only mice receiving TAT-Hsp70-transduced NPCs showed a significantly increased neuronal density (Fig. 3G, 3H). On the other hand, sustained neuroprotection after systemic transplantation of NPCs was observed in animals that had received either native or transduced NPCs. These data suggest that sustained neuroprotection by intracerebral transplantation of NPCs is only achieved via in vitro transduction with TAT-Hsp70, whereas systemic transplantation of NPCs induces long-term neuroprotection independent of cell transduction.
Transplantation of NPCs Reduces Poststroke Neurological Impairment
Neurological deficits in mice were assessed using the rota rod (Fig. 4A, 4B), the tight rope (Fig. 4C, 4D), and the corner turn test (Fig. 4E, 4F). Although intracerebral transplantation of either native or transduced NPCs reduced functional impairment in all behavioral tests as early as day 7, only animals that were treated with TAT-Hsp70-transduced NPCs showed sustained reduced functional deficits. However, systemic injection of either native or transduced NPCs resulted in sustained improved functional outcome in these animals as compared to PBS controls.
Neurogenesis Is Enhanced in Mice Stereotactically Grafted with TAT-Hsp70-Transduced NPCs
Cerebral ischemia was associated with detection of enhanced numbers of proliferating BrdU+ cells for up to 8 weeks within the ischemic hemisphere of each experimental group (Fig. 5A, 5B). Noteworthy, colocalizations of proliferating BrdU+ cells with TUNEL+ cells on day 4 were less than 3% for each experimental condition (representative image in Fig. 5C), suggesting that BrdU+ cells do survive initially. BrdU+ cells were typically located in the SVZ and scattered in the ischemic striatum (Fig. 5D–5N). Systemic transplantation of native or transduced NPCs did not alter the amount of BrdU+ cells within the ischemic hemisphere (Fig. 5A, 5B). However, intracerebral transplantation of TAT-Hsp70-transduced NPCs resulted in a sustained postischemic increase of BrdU+ cells (Fig. 5A, 5B). The latter might be a consequence of either enhanced stimulation of postischemic cell proliferation or enhanced survival of proliferating cells due to intracerebrally grafted TAT-Hsp70-transduced NPCs. In line with this, a differentiation analysis of BrdU+ cells (Fig. 5O–5Z) revealed that only intracerebral transplantation of TAT-Hsp70-transduced NPCs yielded enhanced colocalizations with the immature neuronal marker Dcx and the mature neuronal marker NeuN. On the other hand, systemic transplantation of transduced NPCs did not significantly affect neuronal differentiation of proliferating cells.
NPC-Induced Poststroke Mechanisms Vary with Transplantation Routes
We next analyzed putative mechanisms underlying NPC-mediated neuroprotection after intracerebral and systemic transplantation. Among different events, which contribute to early ischemic injury, MMP-induced breakdown of the blood-brain-barrier (BBB) is one decisive key factor . As such, analysis of MMP gel zymography at 24 hours poststroke (Fig. 6A, 6B) revealed a detection of MMP-9 activity in control animals. However, intracerebral transplantation of neither native nor TAT-Hsp70-transduced NPCs affected MMP-9 activity, whereas systemic injection of NPCs (native/transduced) yielded decreased MMP-9 activity. MMP-2 activity was below detection threshold under all experimental paradigms (Fig. 6A).
Further analysis revealed that BBB integrity was significantly enhanced after systemic application of native/transduced NPCs at 24 hours poststroke (Fig. 6C). Moreover, measurement of TBARS formation at 24 hours showed fewer TBARS in mice systemically treated with native/transduced NPCs (Fig. 6D). Intracerebral injection of NPCs had, however, no effect on TBARS formation. Analysis of long-term effects like astroglial scar formation revealed that scar formation was independent from transduction states of systemically transplanted cells; both native and transduced NPCs reduced glial scar formation (Fig. 6E). On the contrary, only TAT-Hsp70-transduced NPCs mediated reduced glial scar formation after intracerebral transplantation.
Since transplanted NPCs might affect sustained changes within the ischemic lesion site via by-stander effects, we analyzed intracerebral contents of growth factors on day 4 (Fig. 6F) and on day 56 (Fig. 6G). Whereas systemic transplantation of native/transduced NPCs did not significantly affect growth factor contents, intracerebral transplantation of NPCs (native/transduced) increased contents of BDNF, VEGF, and GDNF on day 4. No significant difference between the various intracerebral treatment groups was observed at that time point. On the contrary, growth factor levels on day 56 were significantly increased only in mice treated with intracerebral transplantation of TAT-Hsp70-transduced NPCs. These results suggest that NPC-mediated beneficial effects against stroke depend on the transplantation route chosen.
The efficacy of stem and progenitor cell-based therapies is well established and has already lead to clinical trials [13, 28, 29, 31, 48–50]. In this context, NPCs that can differentiate into both glia and neurons might be advantageous, albeit neural cell replacement seems to be not a prerequisite for cell-based stroke therapy . Nevertheless, therapeutic approaches are limited due to low survival of transplanted cells. We therefore transduced adult NPCs in vitro with the chaperone TAT-Hsp70 in order to enhance resistance of grafted cells within the postischemic milieu. Bearing in mind that the best cell delivery route still remains unknown, NPCs were injected either systemically or intracerebrally. Our data show that sustained neuroprotection after intracerebral transplantation of NPCs correlates with increased survival of grafted cells due to TAT-Hsp70 transduction. On the other hand, systemic transplantation of NPCs mediates acute neuroprotection via different mechanisms, which may include stabilization of the BBB and reduction of ROS despite low intracerebral numbers of grafted cells.
Cell-penetrating peptides like TAT-Hsp70 are an elegant tool to deliver cargo across intact biological membranes . In this context, we have previously shown that in vitro transduction of NPCs with TAT-Bcl-xL is efficient and results in sustained neuroprotection against stroke after intracerebral transplantation of TAT-Bcl-xL-transduced NPCs . Although Hsp70-induced neuroprotection has been shown before [10, 52–54], this study shows for the first time that the fusion protein TAT-Hsp70 enhances resistance of NPCs against hypoxic-hypoglycemic injury. Since neuroprotection by the TAT domain itself has been described in vitro recently [55, 56], we used TAT-HA as a negative control in order to exclude effects of the TAT domain itself on cell viability, neurosphere formation rates, NPC numbers, and differentiation patterns of NPCs.
Although the number of transplanted NPCs gradually declined under each experimental condition, intracerebral cell delivery always yielded higher intracerebral NPC numbers than systemic cell delivery. However, intracerebral transplantation of only TAT-Hsp70-transduced NPCs resulted in significantly enhanced cell numbers within the ischemic striatum for up to 8 weeks when compared with controls. Despite the fact that transplanted NPCs were not exposed to cerebral ischemia itself, secondary cell death of transplanted NPCs in the process of a proinflammatory and proapoptotic ischemic milieu has already been described before [18, 22]. Therefore, enhanced numbers of grafted NPCs after TAT-Hsp70 transduction are most likely due to the antiapoptotic properties of the fusion protein itself.
Analysis of brain injury and functional impairment after intracerebral transplantation of NPCs correlated with the amount of grafted cells within the peri-infarct area. As such, only animals that had been transplanted with TAT-Hsp70-transduced NPCs showed sustained neuroprotection that was associated with improved motor coordination. On the other hand, systemic delivery of native and transduced NPCs significantly reduced brain injury and motor coordination deficits at any time point analyzed, albeit only a small number of scattered NPCs were found within the ischemic hemisphere.
Although stem cell transplantation extends the therapeutic time window as compared to thrombolysis, the optimal time point for transplantation is still elusive and depends on the focus of the therapeutic aim. If reduction of brain injury and infarct volume is to be achieved, acute cell delivery of cells might be most critical . Consequently, we have chosen a 6-hour time window for this study. On the other hand, manipulation of endogenous repair mechanisms such as neuroplasticity might allow cell transplantation at subacute or even later time points [8, 29, 57, 58]. In this context, different cell delivery routes also affect the time window for transplantation. Whereas systemic transplantation of NPCs could benefit from cell homing via inflammation, the latter might induce cell death after local cell transplantation pointing toward different mechanisms by which systemic and intracerebral transplantation of NPCs reduce postischemic brain injury.
Systematic studies on NPC-induced neuroprotection against cerebral ischemia after both systemic and local transplantation do not exist apart from the aforementioned works [33, 34]. Since our study indicates that systemic transplantation of native and transduced NPCs resulted in sustained neuroprotection despite low intracerebral numbers of grafted cells, NPC-induced modulation of extracerebral injurious mechanisms might be intriguing. Among mechanisms leading to acute poststroke brain injury, breakdown of the BBB is one key factor. This study shows that systemic transplantation of NPCs induces a reduced formation of ROS during reperfusion and a reduction of BBB leakage. Along with this, activation of MMP-9, which is critically involved in BBB breakdown , was significantly reduced in animals that had received either native or TAT-transduced NPCs. Nevertheless, one has to keep in mind that a 45-minute stroke induces relatively small infarct sizes that affect both the striatum and parts of the dorsolateral cortex resulting in rather mild breakdown of the BBB .
Intracerebral transplantation of NPCs did not affect BBB leakage or formation of ROS as compared to systemic transplantation of NPCs. Rather, transplantation of TAT-Hsp70-transduced NPCs resulted in sustained enhancement of poststroke neurogenesis as suggested by expression of the neuronal markers Dcx and NeuN in BrdU+-proliferating cells. However, TAT-Hsp70 itself does neither induce neuronal differentiation of SVZ-derived NPCs in vitro nor affect differentiation of grafted cells in vivo. Thus, enhanced poststroke neurogenesis as observed by differentiation analysis of BrdU+ cells is most likely due to the antiapoptotic properties of the fusion protein itself. In other words, TAT-Hsp70 protects intracerebrally transplanted NPCs from secondary cell death resulting in sustained neuroprotection. Taken into account that general mature neuronal differentiation rates of BrdU+ cells after intracerebral transplantation of TAT-Hsp70-transduced NPCs were low with no mature neuronal phenotype of exogenous NPCs observable, neuronal cell replacement or integration of grafted cells within the neural network is not likely. Rather, indirect by-stander effects orchestrating neurorestorative responses of the ischemic milieu might be responsible for NPC-induced neuroprotection after intracerebral transplantation. As such, intracerebral transplantation of TAT-Hsp70-transduced NPCs was associated with increased levels of growth factors after 8 weeks poststroke, possibly being critically involved in NPC-mediated long-term neuroprotection and stimulation of endogenous neurogenesis as observed by enhanced numbers of new-born mature neuronal cells. Enhanced secretion of growth factors is also likely to be involved in mediation of subacute neuroprotection after intracerebral transplantation of NPCs, where growth factor levels were always significantly increased independent of transduction states. Increased growth factor levels on day 4 after intracerebral transplantation correlated with intracerebral cell numbers, which were at that time point still high under each experimental condition. On the contrary, reduction of growth factor contents after intracerebral transplantation of native or TAT-HA-transduced NPCs 8 weeks poststroke is likely due to secondary cell loss of these nontransduced NPCs. Therefore, it is well conceivable that intracerebrally grafted NPCs act via indirect by-stander mechanisms as discussed by us and others [7, 8, 17, 19, 20], for which sufficiently high numbers of residing grafted cells as induced by TAT-Hsp70 are a prerequisite. Nevertheless, a causal relation between growth factor secretion and NPC-mediated neuroprotection still has to be established. On the other hand, systemic transplantation of NPCs mediates acute neuroprotection from the luminal side of the vessels for which high intracerebral cell numbers seem to be not necessary. This observation is in line with previous reports where systemic transplantation of stem and precursor cells initiated beneficial effects despite low cell numbers or even no detectable cells within the ischemic brain [60–63].
This work shows that TAT-Hsp70 successfully enhances survival of NPCs after intracerebral transplantation culminating in long-term neuroprotection against cerebral ischemia. Although cell dosages for intracerebral transplantation did not vary within this study, it is intriguing that the effects seen after intracerebral transplantation depend on high intracerebral numbers of grafted cells. On the other hand, systemic NPC delivery initiates sustained neuroprotection despite low intracerebral numbers of grafted cells via different mechanisms, like stabilization of the BBB and reduction of ROS during early reperfusion. Therefore, systemic NPC delivery might be more feasible for clinical stroke concepts because of its simplicity and due to lower intracerebral cell numbers needed, lacking a need of in vitro manipulation of NPCs such as TAT-Hsp70 transduction. Our study points out the need of a more adequate recognition of NPC actions related to cell delivery routes.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
G.P.H.D. is currently affiliated with the Department 851, Neurodegeneration II, H. Lundbeck A/S, Valby, Denmark. No conflict of interest results from this employment. The remaining authors have nothing to disclose and also have no conflict of interest.