Experimental Neurology 248 (2013) 369–380
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Genetically modiﬁed mesenchymal stem cells (MSCs) promote axonal regeneration and prevent hypersensitivity after spinal cord injury Gentaro Kumagai a,d, Pantelis Tsoulfas a, Satoshi Toh d, Ian McNiece b,1, Helen M. Bramlett a,c, W. Dalton Dietrich a,⁎ a
Department of Neurological Surgery, The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA Interdisciplinary Stem Cell Institute, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA Bruce W. Carter Department of Veterans Affairs Medical Center, Miami, FL, USA d Department of Orthopedic Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan b c
a r t i c l e
i n f o
Article history: Received 19 March 2013 Revised 10 June 2013 Accepted 28 June 2013 Available online 12 July 2013 Keywords: Spinal cord injury Rat bone marrow stromal cells Viral vector transduction MNTS1 p75 Transplantation Axonal regeneration Functional recovery
a b s t r a c t Neurotrophins and the transplantation of bone marrow-derived stromal cells (MSCs) are both candidate therapies targeting spinal cord injury (SCI). While some studies have suggested the ability of MSCs to transdifferentiate into neural cells, other SCI studies have proposed anti-inﬂammatory and other mechanisms underlying established beneﬁcial effects. We grafted rat MSCs genetically modiﬁed to express MNTS1, a multineurotrophin that binds TrkA, TrkB and TrkC, and p75NTR receptors or MSC-MNTS1/p75− that binds mainly to the Trk receptors. Seven days after contusive SCI, PBS-only, GFP-MSC, MSC-MNTS1/GFP or MSC-MNTS1/p75−/GFP were delivered into the injury epicenter. All transplanted groups showed reduced inﬂammation and cystic cavity size compared to control SCI rats. Interestingly, transplantation of the MSC-MNTS1 and MSC-MNTS1/p75−, but not the naïve MSCs, enhanced axonal growth and signiﬁcantly prevented cutaneous hypersensitivity after SCI. Moreover, transplantation of MSC-MNTS1/p75− promoted angiogenesis and modiﬁed glial scar formation. These ﬁndings suggest that MSCs transduced with a multineurotrophin are effective in promoting cell growth and improving sensory function after SCI. These novel data also provide insight into the neurotrophin-receptor dependent mechanisms through which cellular transplantation leads to functional improvement after experimental SCI. © 2013 Elsevier Inc. All rights reserved.
Introduction Mesenchymal stem cells (MSCs) have been reported to repair various organ tissues and prevent immune cell activation and proliferation (Mundra et al., 2013; Nair and Saxena, 2013; Stagg and Galipeau, 2013; Figueroa et al., 2012; English et al., in press). The immunomodulatory characteristics have been demonstrated in a variety of conditions including organ transplantation, autoimmune disease and tissue repair (Mundra et al., 2013). The beneﬁcial effects of exogenous MSCs have also been reported in several clinical conditions including wound healing (Maxson et al., 2012), ischemic–reperfusion injury (Souidi et al., 2013) preservation of intervertebral disc function (Huang et al., 2013) and myocardial regeneration (Przybyt and Harmsen, 2013; Zhang et al., 2013). The safety and therapeutic features of MSCs have also been established for several neuronal diseases including Amyotrophic
⁎ Corresponding author at: Department of Neurological Surgery, University of Miami, Leonard M. Miller School of Medicine, Lois Pope LIFE Center, 1095 NW 14th Terrace, Suite 2-30, Miami, FL 33136-1060, USA. Fax: +1 305 243 3207. E-mail address: [email protected]
(W.D. Dietrich). 1 Current address: Department of Medicine, MD Anderson Cancer Center, Houston, TX, USA. 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.06.028
Lateral Sclerosis, Alzheimer's and Parkinson's Disease, Multiple Sclerosis, Stroke and Traumatic Brain Injury (Laroni et al., in press). Cellular transplantation strategies may also protect and promote regeneration after spinal cord injury (SCI) (Bhanot et al., 2011; Cummings et al., 2005; Frolov and Bryukhovetskiy, 2012; Fujimoto et al., 2012; Hofstetter et al., 2005; Iwanami et al., 2005; Ogawa et al., 2002; Oh et al., 2012; Okada et al., 2005; Rossi and Keirstead, 2009; Sandner et al., 2012; Snyder and Teng, 2012). MSCs have been shown to regulate the immune response after tissue injury and promote functional recovery after SCI (Chopp et al., 2000; Cizkova et al., 2006; Himes et al., 2006; Hofstetter et al., 2002; Karamouzian et al., 2012; Park et al., 2010; Singer and Caplan, 2011; Wu et al., 2003). Recent ﬁndings indicate that MSCs foster host axons to grow into the grafted spinal cord (Ankeny et al., 2004; Hofstetter et al., 2002; Lu et al., 2005). Embryonic and induced pluripotent stem cells are reported to differentiate into neurons, astrocytes, and oligodendrocytes and improve motor function after SCI (Keirstead et al., 2005; Kumagai et al., 2009; Nakamura et al., 2012; Tsuji et al., 2010, 2011). Adult bone marrow MSCs are also capable of self-renewal and differentiation into different mature lineages of mesodermal origin (Grove et al., 2004; Vaquero and Zurita, 2009). Although some studies have suggested that MSCs can differentiate into neural phenotypes after SCI (Akiyama et al., 2002; Alexanian et al., 2011; Chopp et al.,
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2000; Hofstetter et al., 2002), this possibility is presently being debated in the literature (Alexanian et al., 2011; Lu and Tuszynski, 2005; Wright et al., 2011). Neurotrophic factors are known to have beneﬁcial effects on neuroprotective and reparative processes (Conte et al., 2003; Dechant and Neumann, 2002) and may augment regeneration (Longhi et al., 2004; Lu and Tuszynski, 2005). Several approaches have been reported to successfully deliver neurotrophic factors after SCI including the transplantation of genetically modiﬁed cells such as glial restricted progenitors, Schwann cells and MSC transplants (Blits et al., 2005; Cao et al., 2005; Koda et al., 2007; Lu et al., 2005; Shang et al., 2011; Zhang et al., 2010). Neurotrophins exert most of their positive effects, neuronal survival and axonal growth, through the Ras/MAPK and PI3K/Akt signaling pathways by activating the Trk receptors (Huang and Reichardt, 2003). Furthermore, neurotrophins also bind the p75NTR, a member of the tumor necrosis factor receptor (TNRF) superfamily (Dechant and Barde, 2002; Teng et al., 2010). The pro-neurotrophin forms can activate p75NTR, and trigger apoptosis during development or after injury (Teng et al., 2010). In addition, this receptor can serve as a co-receptor for various ligands with different biological activities (Teng et al., 2010). For example p75NTR, interacts with NgR and Lingo-1 (Mi et al., 2004; Wang et al., 2002) and has been reported to exhibit growth inhibitory effects by modulating Rho GTPases (Dubreuil et al., 2003; Yamashita et al., 1999, 2002). Therefore, it is imperative to use neurotrophins that interact mainly with the Trk receptors to eliminate the possibility of these molecules to interact with p75NTR. For this purpose we have made use of a multineurotrophin that can bind to all of the Trk receptors and therefore be able to target diverse neuronal populations that might respond to neurotrophins and created mutations to residues R113 and K114 to reduce the binding to p75NTR (Urfer et al., 1994). In the present study, we grafted MSCs genetically modiﬁed to express MNTS1 or MNTS1/p75− following SCI. Although all transplanted groups attenuated acute inﬂammation and reduced cystic cavity, the transplantation of the MSC-MNTS1 and MSC-MNTS1/p75− but not MSCs alone, promoted axonal growth and prevented cutaneous hypersensitivity after SCI. Moreover, transplantation of MSC-MNTS1/p75− promoted angiogenesis and modiﬁed glial scar formation. These ﬁndings indicate that MSC-MNTS1 are beneﬁcial for improving sensory function, and support a novel cellular transplantation strategy that targets a clinically relevant quality of life issue. Materials and methods Construction of the MNTS1 and MNTS1/p75− Lentivirus The MNTS1 and MNTS1/p75− cDNAs were produced synthetically by Geneart and subcloned into the lentiviral vector pRRLsin.PPT.Th. CMV.MCS.Wpre described earlier (Supplemental Fig. 1) (Dull et al., 1998). For lentiviral production, we used the four plasmid method as described previously (Follenzi and Naldini, 2002). The virus was concentrated by ultracentifugation and stored at −80 °C in the presence of PBS and 0.5% bovine albumin for long term storage. The titers, shown as transducing units (TU) from 108 to 109 TU/ml, were derived using ELISA for the HIV Gag capsid protein p24. The making of MNTS1 has been described earlier (Urfer et al., 1994) and consists of the Human NT-3 with aspartic residue 15 of mature human NT-3 mutated to alanine and the ﬁrst 5 amino acids of the amino terminous changed to the corresponding amino acids of NGF. The MNTS1 binding afﬁnity to the Trk receptor and biological activity has been described in detail (Urfer et al., 1994). The MNST1/p75− was made by changing residues Arg113 and K114 of MNTS1 to alanine. Previously we (Urfer et al., 1994) demonstrated that these amino acids are essential for NT-3 binding to p75NTR. A more detailed explanation about these mutations and on the interaction between p75NTR and NTs are given by Enomoto et al. (2013). Since the MNTS1 backbone is based on NT-3 we expect that these two mutations will reduce the afﬁnity towards p75NTR the same way as the NT-3.
Primary rat marrow stromal cell culture MSCs were collected from femurs and tibias of adult GFP expressing transgenic male Fisher rats. Rats were euthanized with a mixture of 70% CO2 and 30% O2. Tibias and femurs were placed on ice in MEM with alpha modiﬁcation (α-MEM; GIBCO/BRL) containing 20% FCS (Atlanta Biologicals, Norcross, GA), 2 mM L-glutamine (GIBCO/BRL), 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 ng/ml amphotericin B (penicillin, streptomycin, and amphotericin; GIBCO/BRL). Epiphyses of femurs and tibias were removed, and the marrow was ﬂushed out by using a syringe ﬁlled with medium. Bone marrow was ﬁltered through a 70 μm nylon mesh and plated in 75 cm2 ﬂasks. About 24 h after plating, supernatant containing nonadherent cells were removed and fresh medium was added. After the cells had grown to near conﬂuency, they were passaged two to ﬁve times by being detached (0.25% trypsin/1 mM EDTA for 5 min) and replated at a density of ~5000 cells/cm2. Infection of MSCs Before transplantation, MSCs were thawed and grown for 2 days before infecting with the lentivirus. Only 30% of the transgenic MSC cells express GFP. Therefore, we co-infected these cells with a lentivirus expressing GFP. The MSCs did not begin to express GFP until approximately 24 h after infection. LV/GFP/MNTS1 and LV/GFP/MNTS1/p75− infected MSCs exhibited robust transduction efﬁciency with GFP expression in greater than 70% of the cells when we use an MOI of 50. The titer for both LVs was 1 × 109 TU/ml. LV/GFP transduced MSCs continued to express GFP after several passages in culture, and expression was similar in both LV/GFP/MNTS1 and LV/MNTS1/p75− MSCs cultures. Spinal cord injury model Adult female Fischer rats (180–200 g) (n = 48) were housed two per cage and provided food and water ad libitum. All animal procedures and care were approved by the University of Miami Animal Care and Use Committee, in accordance with NIH and the Guide for the Care and Use of Animals. The animals' backs were shaved and cleaned with betadine. For injury and implantation procedures, animals were anesthetized with 1% isoﬂurane and a mixture of 30% oxygen. Following anesthesia, T7–T9 vertebrae were exposed and a dorsal laminectomy was performed at the T8 level to expose the dura. The T7 and T9 vertebrae were held with stabilization clamps. The moderate contusion was created by the Inﬁnite Horizon impactor (200 kdyn; Precision Systems, Lexington, KY) onto the exposed cord. The contusion impact actual force (kdyn), displacement and velocity were monitored. Animals were excluded immediately when actual force errors exceeded 6%. Muscles were subsequently sutured together and the skin was closed using Michel clips. The rats then received intramuscular gentamicin (5 mg/kg) for 7 days and 6 ml of Ringer's solution subcutaneously. The analgesic, Buprenex (Reckitt Benckiser, Richmond, VA; 0.01 mg/kg of 0.3 mg/ml), was administered postsurgery and daily subcutaneously for 2 days. Animals were returned to their cages, and allowed access to food and water along with a heating pad. Temperature and hydration status were carefully monitored for 24 h after injury. Bladders were expressed twice a day for 3–7 days until spontaneous voiding began. MSC transplantation Seven days after injury, the contusion site was re-exposed. Subacute phase, between the acute and chronic phases, is marked by the minimal expression of cytokines, and is likely to be amenable to transplantation therapy (Bhanot et al., 2011; Cummings et al., 2005; Frolov and Bryukhovetskiy, 2012). Before the implantation day, frozen MSCs were thawed into 100-mm dishes and incubated at 37 °C. Primary MSCs at 80–90% conﬂuence were trypsinized from 100-mm dishes,
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twice pelleted at 1500 rpm for 5 min and washed and resuspended in αMEM with 20% FBS. MSCs were collected the morning of the scheduled surgery, kept on ice and implanted within 3 h. Animals were randomly divided into four implant groups: PBS-only, GFP-MSCs (MSCs), MNTS1/GFP-containing LV vector-transduced MSC (MSCMNTS1), and MNTS1/p75−/GFP-containing LV vector-transduced MSC (MSC-MNTS1/p75−). One injection of 6 μl MSCs (4 × 105 cells) was performed over 3 min into the injury epicenter at the midline of the spinal cord at a 90° angle to the cord using a 10-μl Hamilton syringe with a ﬁtted glass capillary tube (112 μm inner diameter). The syringe was pulled out slowly over a period of 3 min. All remaining cells used for transplants showed viabilities of ~95%. Post-operative procedures were performed as described above. Behavioral assessment Two non-biased observers analyzed hindlimb performance using the Basso, Beattie and Bresnahan (BBB) scoring system (Basso et al., 1995). A BBB sub-score also was obtained because some animals did not exhibit the same progression of functional improvements as delineated by the BBB score (Pearse et al., 2004). All animals were subjected to BBB testing once a week from injury until sacriﬁce. Cutaneous sensitivity to heat and mechanical stimuli were evaluated. First, rats were placed in Plexiglas containers that rested on an elevated glass surface. A mobile infra-red emitter below the glass was placed under the center of the rat's plantar hind paw and activation of the emitter started a timer (Hargreaves et al., 1988). A hind paw withdrawal from the glass stopped the emitter and timer. The duration between activation of the infra-red stimulus and its termination was the withdrawal latency (measured in seconds) with mean of three measures reported as the ﬁnal withdrawal latency. After latency determination, rats were placed in Plexiglass containers that rested on an elevated wire mesh. Hind paw withdrawal thresholds (measured in grams) to an innocuous mechanical stimulus were measured using a set of von Frey ﬁlaments (Chaplan et al., 1994). All animals were subjected to sensory testing 4 and 6 weeks from injury until sacriﬁce. Histological analysis Six weeks after injury (5 weeks post-implantation), animals were anesthetized using 3% isoﬂurane, 70% N2O and 30% O2 transcardially perfused with 300 ml of cold (approximately 4 °C) 0.9% NaCl solution, followed by 500 ml of phosphate-buffered 4% paraformaldehyde. Brains and spinal cords were dissected immediately after perfusion and placed in 4% paraformaldehyde overnight. The next day, the ﬁxative was replaced with a solution of 30% w/v sucrose and 0.1% sodium azide. The center of the injury/implant area was identiﬁed, and 6 mm rostral and caudal (12 mm total) were removed and embedded in gelatin (Oudega et al., 1994) overnight at 37 °C. The tissue was next placed in the gelatin block and immersed in 4% paraformaldehyde overnight, followed by 30% w/v sucrose until analysis. A freezing microtome (Leica SM2000R) was used to section the injury/implant area (T6–T10) coronally at 35 μm. The sections were stored at 4 °C in 0.1 M phosphate buffer containing 0.1% sodium azide (pH 7.4). Other spinal cords were sectioned in the sagittal at 30 μm/axial plane at 20 μm on a cryostat (Leica CM1900). The injured spinal cords from the four groups were histologically evaluated by Luxol Fast Blue (LFB) and hematoxylin and eosin (H&E) staining and immunohistochemistry. Tissue sections were stained with the following primary antibodies: anti-GFP (chicken IgY, 1:1000, Aves Labs), Alexa488-conjugated anti-GFP (rabbit IgG, 1:200, Life technologies), anti-GFAP (mouse IgG, 1:1000, BD Pharmigen), anti- Neuroﬁlament Subunit (NF-M, rabbit IgG, 1:2000, EnCor Biotechnology Inc.), anti-RECA-1 (mouse IgG, 1:50, AbD Serotec), anti-ED-1 (mouse IgG, 1:100, Serotec) and anti- Calcitonin Gene-Related Peptides (CGRP, Goat IgG, 1:200, Abcam).
For immunohistochemistry with anti-NF-M, RECA-1, -ED-1, -GFAP, and -CGRP, we used a biotinylated secondary antibody (Jackson Immunoresearch Laboratory, Inc.), after exposure to 0.3% H2O2 for 30 min at room temperature to inactivate endogenous peroxidase. The signals were enhanced with the Vectastain ABC kit (Vector Laboratories, Inc.). Nuclei were stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen). The samples were examined with a universal ﬂuorescence microscope (Axiovert 200 M, Carl Zeiss) or a confocal laser scanning microscope (FV1000, OLYMPUS). Quantitative analyses of stained tissue sections To determine the effects of the various MSC treatments on cavity volume images of LFB/H&E, or immunostained sections, images were obtained by a universal ﬂuorescence microscope (Axiovert 200 M, Carl Zeiss), manually outlined and quantiﬁed by Neurolucida 9 (MBF Bioscience, Inc., Wiliston, VT, USA). Constant threshold values were maintained for all the analyses with Neurolucida 9. Data from remaining normal cord tissue, cavities, total abnormal tissue, and preserved myelin were obtained axially from sections throughout the 12 mm segment (including the epicenter) every 500 μm using a Zeiss Axiovert 200 M microscope (n = 4, each). Volumes were estimated using the planimetric analysis with Neurolucida software; 3D reconstructions were made using Neurolucida software (Hurtado et al., 2012; Oudega et al., 1994). Lesioned cord areas were deﬁned as containing damaged and/or necrotic tissue, altered size and number of neurons and their normal distribution in the gray matter, small cell inﬁltration and cavitation (lack of tissue). Data for lesion size are reported as percentages of contour (total area of cross section) throughout the analyzed 12 mm segment. To determine whether MSC treatment would promote axonal growth, we stereologically quantiﬁed NF-M-positive axonal ﬁbers after spinal cord injury at the epicenter and 4 mm rostral and caudal to the epicenter within each animal using axial sections. The spinal cord was outlined at 5 ×, and then a 100× oil immersion objective. Using Stereoinvestigator software and the optical fractionators sampling design (n = 4, counting frame area, 25 × 25 μm2, sampling grid 200 × 200 μm2). To assess the effects of MSC transplantation on the inﬂammatory cellular response to SCI, ED-1 and GFAP-positive cells were quantiﬁed using stereology of axial sections at the epicenter. The spinal cord was outlined at 5 ×, and then a 63 × oil immersion objective using Stereoinvestigator software and the optical fractionators sampling design (n = 4, counting frame area, 25 × 25 μm2, sampling grid 200 × 200 μm2). To assess angiogenesis after spinal cord injury, RECA1-positive blood vessels were counted manually in axial sections of the lesion epicenter at 20× magniﬁcation (n = 4). Finally, to determine CGRP-positive nerve ﬁber length was calculated after spinal cord injury at the 4 mm caudal to the epicenter, axial sections were quantiﬁed using stereology. The lamina III in the gray matter was outlined at 10×, and then with a 63× oil immersion objective using Stereoinvestigator software and the optical fractionators sampling design (n = 4, sampling grid 75 × 75 μm2). Statistical analysis All data are presented as the mean ± SEM. Depending on the outcome measures, speciﬁc statistical analytical procedures were conducted. One-way ANOVA followed by the Tukey–Kramer test for multiple comparisons across groups was used for RECA-1-, ED-1-, GFAP-, and CGRP-stained analysis. Repeated measures two-way ANOVA followed by the Tukey–Kramer test was used for LFB/H&E, NF-M, BBB score and BBB sub-score analysis. Spearman correlation coefﬁcient was used for correlation of the results of heat and mechanical stimuli analysis and the quantiﬁcation of CGRP-positive ﬁbers length. In all statistical analyses, the signiﬁcance was set at P b 0.05.
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Results Transplanted MSC-MNTS1 or -MNTS1/p75−, but not MSCs reduce cutaneous hypersensitivity We monitored the locomotor functional recovery in all four groups: control, MSC, MSC-MNTS1 and MSC-MNTS1/p75−, respectively using the BBB scoring scale and BBB sub-score (Basso et al., 1995; Pearse et al., 2004) (Figs. 1A and B). The contusive SCI resulted in complete paralysis (BBB score 0) on day 1, followed by gradual recovery with a plateau around 14 days. The mean values of the BBB score on day 42 were 9.1 ± 0.4, 10.3 ± 0.4, 10.4 ± 0.4 and 10.8 ± 0.6, respectively. There was no signiﬁcant difference in the BBB scores between the cell transplant and control groups. The mean values of the BBB sub-score on day 42 were 1.8 ± 0.6, 3.5 ± 0.8, 4.3 ± 1.1 and 3.9 ± 0.9, respectively. Also, there was no signiﬁcant difference in the BBB sub-scores between the cell transplant and control groups. To evaluate the sensitivity of hindlimb cutaneous hypersensitivity, heat and mechanical stimuli were examined at 4 and 6 weeks after injury (Figs. 1C and D). Prior to contusion injury, the mean latency in second(s) to withdraw from a noxious heat stimulus was 17.7 ± 0.4 s (Intact group, n = 10). Following contusion injury, withdrawal latencies to noxious heat in the control and MSC groups were signiﬁcantly (P b 0.05) decreased at 4 or 6 weeks following injury (4 weeks; 12.5 ± 0.8 and 13.4 ± 0.9 s or 6 weeks; 11.7 ± 0.8 and 11.8 ± 0.7 s, respectively). On the other hand, the MSC-MNTS1 and MSC-MNTS1/ p75− group demonstrated signiﬁcantly (P b 0.05) improved withdrawal thresholds compared to the control and MSC groups at 6 weeks after contusion, such that thresholds (6 weeks; 14.6 ± 0.7 and 16.2 ± 0.6 s, respectively) were no different from that of pre-contusion thresholds. Prior to contusion injury, the mean withdrawal threshold to innocuous mechanical stimuli was 14.8 ± 0.2 g (Intact group, n = 10).
Following contusion injury, withdrawal latency to mechanical stimuli in the control and MSC groups was signiﬁcantly (P b 0.05) decreased at 4 or 6 weeks following injury (4 weeks; 8.0 ± 1.2 and 10.2 ± 1.0 s or 6 weeks; 9.1 ± 1.0 and 11.4 ± 0.9 s, respectively). In contrast, the MSC-MNTS1 group demonstrated improved withdrawal thresholds compared to the control group at 4 or 6 weeks after contusion, such that thresholds (4 weeks; 12.1 ± 1.0 s, 6 weeks; 12.9 ± 0.6 s, respectively) were no different from that of pre-contusion thresholds. The MSC-MNTS1/p75− group also demonstrated improved withdrawal thresholds compared to the control and MSC group at 6 weeks after contusion, such that thresholds (14.3 ± 0.5 s) were no different from that of pre-contusion thresholds. Transplanted MSCs, -MNTS1, and -MNTS1/p75− reduce cavity size after SCI but not lesion size or preserved myelin Histopathological analysis of tissue by hematoxylin and eosin and luxol fast blue staining was based on area and volume values of contusion calculated by computer extrapolation using stereological methods from contour/analysis of single sections (Fig. 2A). A well demarcated contusion was observed in all animals. In some cases, 3-D images were created to visualize the extent of damage to the cords in the epicenter and beyond (Fig. 2B). Quantitative data from the epicenter and throughout the injured spinal cord are presented in Fig. 3. Cavity, lesion, and preserved myelin volumes and area were obtained in the control and transplant groups. Cavity volume revealed signiﬁcantly greater preservation in the MSC-MNTS1/p75− group compared with the control group (Fig. 3A, P = 0.036). Cavity area at 1000 μm rostral to the epicenter revealed signiﬁcantly greater preservation in MSC, MSC-MNTS1, and MSC-MNTS1/p75− compared with the control group (Fig. 3B, P = 0.014, P = 0.042, and P = 0.011, respectively). There was no signiﬁcant difference in the lesion volume and area between
Fig. 1. Transplanted MSC-MNTS1 and MSC-MNTS1/p75−, but not MSCs, reduces the sensitivity of cutaneous hypersensitivity. (A and B) Open ﬁeld walking as determined by the BBB scores and BBB sub-score. Downward arrow indicates time of transplantation. Hindlimb function was assigned scores from 0 to 21 (ﬂaccid paralysis to normal gait). Values are means ± SEM (n = 12, each). (C and D) Rats were tested at 4 and 6 weeks following contusion and hind paw responses to Noxious heat (Heat) and innocuous mechanical (Mechanical) stimuli were recorded. Values are means ± s.e.m. (n = 12, each). *: P b 0.05. **: P b 0.01.
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Fig. 2. Cavity, lesion formation, and preserved myelin are evaluated by LFB/HE staining. (A, C, E, G) Representative micrographs of rat spinal cord axially sections at the 1000 μm rostral to the epicenter of the insult. Sections are stained by hematoxylin and eosin and luxol fast blue. Note patterns of cavity and lesion formation in gray and white matter structures of the control (A), MSC (C), MSC-MNTS1 (E), and MSC-MNTS1/p75− (G). Scale bar: 500 μm. (B, D, F, H) 3-D reconstruction of injury patterns in control (B), MSC (D), MSC-MNTS1 (F), and MSC-MNTS1/p75− (H) groups. Notice similar patterns of spinal cord (blue), cavity formation (yellow), lesion (red), and preserved myelin (orange).
the cell transplant and control groups (Figs. 3C and D). There was also no signiﬁcant difference in preserved myelin volume and area between the cell transplant and control groups (Figs. 3E and F).
Transplanted MSC-MNTS1 and -MNTS1/p75− but not MSCs promote axonal growth after SCI To assess the effect of increased neurotrophin on axonal growth by comparing the MSC, MSC-MNTS1, MSC-MNTS1/p75− groups, we examined Neuroﬁlament subunit (NF-M)-positive axons using stereology at the epicenter and at 4 mm rostral and caudal to the epicenter in axial sections (Fig. 4). While few NF-M-positive neuronal axons were observed at the rim of the lesion epicenter in both the control and MSC groups, there were signiﬁcantly (P b 0.05) more NF-M-positive neuronal axonal counts in the MSC- MNTS1/p75− group at the 4 mm site rostral to the lesion epicenter compared with the control group (P = 0.011), and at the lesion epicenter compared with the control and MSC groups (P = 0.020 and P = 0.009, respectively). There were signiﬁcantly more NF-M-positive axonal counts in the MSC-MNTS1 group at the 4 mm caudal to the lesion epicenter site compared with the MSC group (P = 0.043).
Transplanted MSCs, MSC-MNTS1, and -MNTS1/p75− reduce expression of immune cells while MSC-MNTS1/p75− suppress astrocyte reactivity after SCI Contusion injury to the spinal cord results in the chronic inﬂammatory events including activated microglia and macrophages. To assess the effects on the recruitment of these cells in the injury site, we examined ED-1 immuno-positive cells which, are characterized as activated microglia/macrophages using stereology at the epicenter in axial sections (Figs. 6A–D, I). While ED-1 immunoreactivity of the control group was found in both the gray and white matter structures, immunoreactivity in the MSC, -MNTS1, and -MNTS1/p75− groups were reduced in both the gray and white matter (Figs. 6A–D). Quantitative analysis revealed that there were signiﬁcantly more ED1-positive cells at the epicenter site compared with the MSC, -MNTS1, and -MNTS1/ p75− groups (P b 0.01, each). To assess the effects of transplantation on astrocyte reactivity after SCI, axial sections of the injured spinal cord were also examined immunohistochemically using stereology for GFAP (Figs. 6E–H, J). While GFAP immunoreactivities of the control and MSC groups were found in the white matter, those of the MSCMNTS1 and -MNTS1/p75− groups were reduced in that area. There were also signiﬁcantly more GFAP-positive cells in the control group at the epicenter site compared with the MSC-MNTS1/p75− groups (P = 0.037).
Transplanted MSC-MNTS1/p75− but not MSCs and MSC-MNTS1 promote angiogenesis
Transplanted MSC-MNTS1/p75− reduce the expression of CGRP-positive ﬁbers after SCI
To assess the effects of transplantation on the extent of angiogenesis after SCI, axial sections of the injured spinal cord were examined immunohistochemically using stereology for RECA-1 (Fig. 5, a marker for endothelial cells). While a few RECA-1-positive blood vessels were observed at the lesion site in axial sections of the control and MSC groups, signiﬁcantly more RECA-1-positive blood vessels were found in the MSC MNTS1p75− group compared with the control and MSC groups (P b 0.01 and P = 0.024, respectively).
We quantiﬁed calcitonin gene-related peptide (CGRP)-immunoreactive sensory axons using stereology at 4 mm level caudal to the epicenter in axial sections (Fig. 7). The control group gave rise to sprouting of CGRP-positive ﬁbers into Rexed lamina III (Fig. 7A). Quantitative analysis revealed that there were signiﬁcantly more CGRP-positive ﬁbers at the level 4 mm caudal to the epicenter site of control animals compared with the MSC-MNTS1/p75− groups (Fig. 7E, P b 0.01). Importantly, there was a reciprocal relationship between the withdrawal
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Fig. 3. Transplanted MSCs, MSC-MNTS1, and MSC-MNTS1/p75− reduce cavity size after SCI, but did not affect lesion size or preserve myelin. (A, C, and E) Quantitative histopathological assessment of cavity, lesion, and preserved myelin volume percentages in control, MSC, -MNTS1, and -MNTS1/p75− groups; negative signs represent the rostral direction. Cavity volume revealed signiﬁcantly greater preservation in the MSC-MNTS1/p75− group compared with the control group. Values are means ± SEM (n = 4, each). *: P b 0.05, Control vs. MSC-MNTS1/p75−. (B, D, and E) Quantitative histopathological assessment of cavity, lesion, and preserved myelin area percentages across the rostral and caudal regions from epicenter (0.0 μm) in control, MSC, MSC-MNTS1, and MSC-MNTS1/p75− groups; negative signs represent the rostral direction. Cavity Area at the sites 1000 um rostral to the epicenter revealed signiﬁcantly greater preservation in MSC, MSC-MNTS1, and MSC-MNTS1/p75−. There was no signiﬁcant difference in the lesion and preserved myelin volume/area between the cell transplant and control groups. Values are means ± SEM (n = 4, each). *: P b 0.05, Control vs. MSC-MNTS1/p75−, #P b 0.05, Control vs. MSC-MNTS1, †P b 0.05, Control vs. MSC.
threshold to heat and mechanical stimulation of individual rats and the amount of CGRP-positive ﬁbers in all 16 animals including the control and transplanted groups (Figs. 7F and G, P = 0.038 and P b 0.01; Spearman correlation coefﬁcient, −0.522 and −0.636, respectively).
spinal cord were examined immunohistochemically for GFP. At 3 post transplant periods, large numbers of transplanted cells were identiﬁed at 1 week after transplantation, followed by a gradual decrease around 4 weeks (Supplemental Figs. 2A and B). In contrast, few grafted cells were found at 6 weeks after transplantation (Supplemental Fig. 2C).
GFP-positive MSCs, -MNTS1, and -MNTS1/p75− grafted cells do not survive after spinal cord injury
To assess the cell survival of the MSC, -MNTS1, and -MNTS1/p75− grafted onto the injured spinal cord, coronal sections of the injured
Together our results indicate that cellular treatments after contusive spinal cord injury with MSC-MNTS1 or MSC-MNTS1/p75− reduce
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Fig. 4. Transplanted MSC-MNTS1 and -MNTS1/p75−, but not MSCs, promote axonal growth. (A–D) Representative images of axial sections stained for NF-M at the lesion epicenter in all four groups. Many more NF-M-positive axons were present in axial sections of the MSC-MNTS1 (C) and MSC-MNTS1/p75− (D) groups, when compared to control (A), or MSC (B) groups. Scale bar: 50 μm. (E) Quantitative analysis of the NF-M-positive axon counts at the epicenter and 4 mm rostral and caudal to the epicenter. While few NF-M-positive neuronal axons were observed at the rim of the lesion epicenter in the control group, there were signiﬁcantly more NF-M-positive neuronal axon counts in the MSC-MNTS1/p75− group at 4 mm rostral to the lesion epicenter site compared with the control group, and at the lesion epicenter compared with the control and MSC groups. There were signiﬁcantly more NF-M-positive neuronal axon counts in the MSC-MNTS1 group at 4 mm caudal to the lesion epicenter site compared with the MSC group. Values are means ± SEM (n = 4, each). *: P b 0.05, Control vs. MSC-MNTS1 or MSC-MNTS1/p75−. **: P b 0.01, MSC vs. MSC-MNTS1/p75−.
cutaneous hypersensitivity but do not improve recovery of motor function. Although our studies showed that in all transplanted groups the early survival of transplanted cells, attenuation of acute inﬂammation and a reduced cystic cavity volume, the transplantation of the MSCMNTS1 and MNTS1/p75−, but not the naïve MSCs, promoted axonal growth and prevented hypersensitivity after SCI. Moreover, transplantation of MSC-MNTS1/p75− promoted angiogenesis and modiﬁed
glial scar formation. Finally, we provide for the ﬁrst time evidence that treatment with transduced MSCs alleviated SCI-induced mechanical allodynia and thermal hyperalgesia of the hindlimb signiﬁcantly better than documented with naïve MSCs. Recently, transplantation of MSCs after spinal cord injury has been widely tested because they can be used as autografts, readily collected within a short time, and genetically modiﬁed to overexpress
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Fig. 5. Transplanted MSC-MNTS1/p75−, but not MSCs and MSC-MNTS1, promote angiogenesis after SCI. (A–D) Representative images of axial sections stained for RECA-1 at the lesion epicenter in all groups. Many more RECA-1-positive blood vessels were present in axial sections of the MSC-MNTS1/p75− groups (D), when compared to control (A), MSC (B), and MSC-MNTS1 (C) groups. Scale bar: 100 μm. (E) Quantitative analysis of blood vessels at the lesion epicenter. RECA-1 immunostaining revealed that signiﬁcantly more RECA-1-positive vessels were observed at the lesion site in the MSC-MNTS1/p75− group compared with the control and MSC groups. Values are means ± SEM (n = 4, each). *: P b 0.05, Control vs. MSC-MNTS1/p75−. **: P b 0.01, MSC vs. MSC-MNTS1/p75−.
neurotrophic factors. Published studies indicate that the transplantation of MSCs promote overall functional recovery after SCI (Chopp et al., 2000; Cizkova et al., 2006; Hofstetter et al., 2002; Wu et al., 2003) and do not induce allodynia in response to mechanical or thermal stimulation (Abrams et al., 2009). The mechanisms by which MSCs exert their effects on the injured spinal cord have also begun to be elucidated (Parr et al., 2007; Rossi and Keirstead, 2009; Wright et al., 2011).
There is increasing evidence that MSCs may be immunosuppressive, synthesize a number of neurotrophic cytokines stimulating nerve growth, including BDNF, NGF, and vascular endothelial growth factor (VEGF). These transplanted cells may also act as guides for regenerating axons across the lesion site of the injured spinal cord (Wright et al., 2011). In contrast to reported positive ﬁndings, other studies indicate no signiﬁcant improvements on BBB motor score after transplantation of
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Fig. 6. Transplanted MSCs, -MNTS1, and -MNTS1/p75− reduce the chronic expression of immune cells and MSC-MNTS1/p75− suppress astrocyte reactivity after SCI. (A–H) Representative images of axial sections for ED-1 and GFAP at the lesion epicenter in all four groups. ED-1 immunoreactivity was conﬁned to the gray matter of the MSC (B), MSC-MNTS1 (C), and MSC-MNTS1/p75− (D) groups and in the gray and surrounding white matter of the control group (A). GFAP immunoreactivity was less intense in MSC (F), and MSC-MNTS1 (G), and MSC-MNTS1/p75− (H) compared to the control group (E). Scale bar: 500 μm. Black boxes indicated higher-magniﬁcation image of the area. Scale bar: 50 μm in black boxes. (I) Quantitative analysis of the ED-1-positive cell counts at the epicenter. There were signiﬁcantly more ED-1-positive cells in the control group at the epicenter site compared with the MSC, -MNTS1, and -MNTS1/p75− groups. Values are means ± SEM. (n = 4). **: P b 0.01, Control vs. MSC, or MSC-MNTS1, and MSC-MNTS1/p75−. (J) Quantitative analysis of the GFAP-positive cells counts at the epicenter. There were also signiﬁcantly more GFAP-positive cells in the control group at the epicenter site compared with the MSC -MNTS1/p75− group. Values are means ± SEM. (n = 4). *: P b 0.05, Control vs. MSC-MNTS1/p75−.
MSCs into a contused or transected spinal cord (Lu et al., 2005; Park et al., 2010; Wright et al., 2011; Yano et al., 2005; Yoshihara et al., 2006). Here we report that naïve MSC also failed to promote functional recovery and reduce hypersensitivity after contusive SCI. MSCs have been shown to attenuate chronic inﬂammation and injury-induced sensitivity to mechanical stimuli after experimental SCI (Abrams et al., 2009). Our results show that although there were signiﬁcantly less ED1-positive cells in the naïve MSC transplanted group compared with control, no signiﬁcant prevention of hypersensitivity after SCI was observed with this cell treatment. The generation of neuropathic pain after SCI is a major quality of life issue for people living with SCI (Fehlings and Wilcox, 2011; Hama and Sagen, 2012). Regarding the positive results of preventing hypersensitivity in the MSC-MNTS1 and -MNTS1/p75− groups, several mechanisms may underlie the improvement of sensory function (Pezet and McMahon, 2006). Neurotrophins affect essentially all biological aspects of neuronal function, including survival, differentiation, growth and apoptosis by using a two-receptor system, consisting of Trk tyrosine
kinases and the p75NTR neurotrophin receptor (Kaplan and Miller, 2000). Three types of Trk receptors, namely TrkA, TrkB and TrkC mediate the biological activity of neurotrophins. Trk receptor tyrosine kinases undergo rapid trans-phosphorylation following ligand binding, leading to a cascade of protein phosphorylation in the cell. The distinctive neuronal deﬁciencies reported in mice with null mutations of the TrkA, TrkB and TrkC genes are similar to those observed in mice with null mutations in the NGF, BDNF and NGF genes, respectively. Therefore, the Trk receptor appears to mediate the survival promoting action of neurotrophins on developing neurons. Several approaches have been reported to deliver genetically modiﬁed MSC transplants into the injured spinal cord (Koda et al., 2007; Lu et al., 2005; Shang et al., 2011; Zhang et al., 2010). After transduction to express high quantities of BDNF, the density of CGRP-labeled sensory and ChAT-labeled motor axons increased after spinal cord transection (Lu et al., 2005). Another study reported that adenovirus vector-mediated ex vivo gene transfer of BDNF promoted axonal regeneration in a transection model (Koda et al., 2007). These
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Fig. 7. Transplanted MSC-MNTS1/p75− reduce the expression of CGRP-positive ﬁbers after SCI. (A–D) Representative images of axial sections for CGRP-positive ﬁbers 4 mm caudal to the epicenter in all four groups. More CGRP-positive ﬁbers were observed in lamina III of control group (A) compared with MSC (B), MSC-MNTS1 (C), and MSC-MNTS1/p75− (D). Arrows indicate CGRP-positive ﬁbers. Scale bar: 100 μm. (E) Quantitative analysis of the CGRP-positive ﬁbers at 4 mm caudal to the epicenter. There were signiﬁcantly more CGRP-positive ﬁbers in the Control group compared with the MSC-MNTS1/p75− group. Values are means ± SEM. (n = 4). **: P b 0.01, Control vs. MSC-MNTS1/p75−. (F, G) The amount of CGRP-positive ﬁbers in lamina III correlates with the withdrawal threshold of heat (F) and mechanical stimuli in all 16 animals including the control and transplanted groups (G). Black circles indicate the control and MSC groups. White circles indicate the MSC-MNTS1 and -MNTS1/p75− groups.
cells also enhanced axonal regeneration of TH-positive coerulospinal ﬁbers at the injury level and CGRP-positive sensory ﬁbers at the caudal level of the cellular graft. Multineurotrophin NT3/D15A is capable of mimicking the downstream effects of BDNF and NT-3 through binding to both TrkB and TrkC receptors, respectively (Urfer et al., 1994). Transplantation of NT3/D15A expressing glial-restricted precursor cells in the subacute phase of SCI led to an increase of myelin formation and recovery of hindlimb locomotion (Cao et al., 2005). Transplantation of NT3/D15Aexpressing Schwann cells in the subacute phase of SCI also promoted the survival of transplants and increased myelination as well as 5-HT, DβH-, and CGRP-positive ﬁbers in the implants (Golden et al., 2007). MNTS1, which contains only seven amino acid changes, binds all receptors of the Trk family. This molecule efﬁciently induces autophosphorylation of TrkA, TrkB and TrkC and supports the survival of more sensory neurons from DRG and NG as well as sympathetic neurons than any of the neurotrophins individually (Urfer et al., 1994). Transduction with MNTS1 is therefore a novel way to combine MSC transplantation with a growth factor that activates TrkA, TrkB, and TrkC receptors to improve repair after SCI. Although transplanted MSCMNTS1 and -MNTS1/p75−, but not MSCs promoted NF-M-positive ﬁbers in the present study, they did not promote CGRP-positive ﬁbers. CGRP-immunoreactive dorsal root neurons express the nerve growth factor receptor TrkA (Averill et al., 1995) and increase both neuropeptide expression and sprouting with elevated NGF levels (Christensen and Hulsebosch, 1997). CGRP is expressed in small- to mediumdiameter DRG neurons (Hokfelt et al., 1992) which are involved in pain transmission, temperature, and noxious and non-noxious mechanical stimuli. Blocking of CGRP attenuates allodynia of the forelimbs in spinal cord models of chronic pain (Bennett et al., 2000). On the other hand, it is reported that the beneﬁcial effects of MSC-derived astrocytes
are limited by graft-induced allodynia in an experimental model of spinal cord injury (Hofstetter et al., 2005). In that study, the transduction of NSCs with neurogenin-2 before transplantation suppressed astrocytic differentiation of engrafted cells and prevented graft-induced sprouting of the CGRP-immunoreactive ﬁbers into Rexed lamina III and allodynia. In our study, in addition to reducing posttraumatic inﬂammation and the suppression of CGRP-immunoreactive ﬁbers in Rexed Lamina III may have led to the observed improvements in sensory responses after SCI. One of the most prominent biological functions of p75NTR is that it induces cell death through the death-domain sequence which is distantly related to the intracellular domains of Fas and TNF receptors. This effect is mediate by the proneurotrophin form (Kaplan and Miller, 2000). Neurotrophins and their receptors are essential growth factors in the formation of the heart and vascular system and the complete ablation of the p75NTR causes severe developmental defects (von Schack et al., 2001). Also, p75NTR has been implicated in endothelial cell apoptosis (Caporali et al., 2008). Consistent with these ﬁndings, our study shows that animals receiving MSCs secreting the MNTS1/ p75− had more RECA-1-positive blood vessels within the injured spinal cord. This result may be related to the Trk receptor activation which has been shown to promote angiogenesis. Although we observed reduced cavity volume and extensive axonal growth in the transplanted MSC-MNTS1 and -MNTS1/p75− groups, this morphological ﬁnding did not result in improved locomotor functional recovery. The failure to observe motor recovery is most likely due to the fact that the cell transplants may not have survived chronically in the present contusive SCI model. Thus, longer term survival may be necessary for the cell transplants to inﬂuence the preservation of myelin and vital white matter tracts critical in the return of locomotor function after thoracic SCI. Also, additional studies are required to determine whether
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multiple MSC injections at possibly different pericontusional sites may be necessary to promote walking in this SCI model. In summary, MSCs have a great potential as therapeutic agents against various human diseases including SCI. These cells can be readily obtained through established clinical procedures, are easy to isolate and expand for autotransplantation with limited risk of rejection. In this study, we have succeeded in preventing cutaneous hypersensitivity after SCI by the transplantation of MSCs secreting a multineurotrophin without p75 activity. Further studies are required to ensure the longterm safety and efﬁcacy of manipulated MSCs to improve locomotor function and other quality of life issues after SCI. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.expneurol.2013.06.028. Acknowledgments We thank Alexander Marcillo, Ramon German, Eva Juarez, Christina Gutierrez, Iliana Oropesa, Shimin Lin for the animal care and behavioral testing; Dr Beata Frydel, David Sequeira for the histological analysis; and Haruo Kanno, Ofelia Furones-Alonso, Abdul Dawood, Yungang Wang for technical assistance, and all the members of Dr. Dietrich's laboratory for encouragement and kind support. We also thank Drs. K. Ueyama, S. Toh, Y. Ishibashi, and A. Ono for encouragement and kind support. Funding was provided by the USAMRMC U.S. Army Medical Research Command, Battleﬁeld Exercise and Combat Related Spinal Cord Injury Program (W81XWH1010737), the Miami Project to Cure Paralysis and Veterans Affairs 1 I01 BX000521. Dr. Gentaro Kumagai is currently at Department of Orthopedic Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan. Disclosure of potential conﬂict of interests The authors declare no competing ﬁnancial interest or conﬂict of interest. References Abrams, M.B., Dominguez, C., Pernold, K., Reger, R., Wiesenfeld-Hallin, Z., Olson, L., Prockop, D., 2009. Multipotent mesenchymal stromal cells attenuate chronic inﬂammation and injury-induced sensitivity to mechanical stimuli in experimental spinal cord injury. Restor. Neurol. Neurosci. 27, 307–321. Akiyama, Y., Radtke, C., Kocsis, J.D., 2002. Remyelination of the rat spinal cord by transplantation of identiﬁed bone marrow stromal cells. J. Neurosci. 22, 6623–6630. Alexanian, A.R., Fehlings, M.G., Zhang, Z., Maiman, D.J., 2011. Transplanted neurally modiﬁed bone marrow-derived mesenchymal stem cells promote tissue protection and locomotor recovery in spinal cord injured rats. Neurorehabil. Neural Repair 25, 873–880. Ankeny, D.P., McTigue, D.M., Jakeman, L.B., 2004. Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats. Exp. Neurol. 190, 17–31. Averill, S., McMahon, S.B., Clary, D.O., Reichardt, L.F., Priestley, J.V., 1995. Immunocytochemical localization of trkA receptors in chemically identiﬁed subgroups of adult rat sensory neurons. Eur. J. Neurosci. 7, 1484–1494. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open ﬁeld testing in rats. J. Neurotrauma 12, 1–21. Bennett, A.D., Chastain, K.M., Hulsebosch, C.E., 2000. Alleviation of mechanical and thermal allodynia by CGRP(8-37) in a rodent model of chronic central pain. Pain 86, 163–175. Bhanot, Y., Rao, S., Ghosh, D., Balaraju, S., Radhika, C.R., Satish Kumar, K.V., 2011. Autologous mesenchymal stem cells in chronic spinal cord injury. Br. J. Neurosurg. 25, 516–522. Blits, B., Kitay, B.M., Farahvar, A., Caperton, C.V., Dietrich, W.D., Bunge, M.B., 2005. Lentiviral vector-mediated transduction of neural progenitor cells before implantation into injured spinal cord and brain to detect their migration, deliver neurotrophic factors and repair tissue. Restor. Neurol. Neurosci. 23, 313–324. Cao, Q., Xu, X.M., Devries, W.H., Enzmann, G.U., Ping, P., Tsoulfas, P., Wood, P.M., Bunge, M.B., Whittemore, S.R., 2005. Functional recovery in traumatic spinal cord injury after transplantation of multineurotrophin-expressing glial-restricted precursor cells. J. Neurosci. 25, 6947–6957. Caporali, A., Pani, E., Horrevoets, A.J., Kraenkel, N., Oikawa, A., Sala-Newby, G.B., Meloni, M., Cristofaro, B., Graiani, G., Leroyer, A.S., Boulanger, C.M., Spinetti, G., Yoon, S.O., Madeddu, P., Emanueli, C., 2008. Neurotrophin p75 receptor (p75NTR) promotes endothelial cell apoptosis and inhibits angiogenesis: implications for diabetesinduced impaired neovascularization in ischemic limb muscles. Circ. Res. 103, 15–26. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., 1994. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63.
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