Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

Cytotherapy, 2010; 12: 792–806 Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spi...

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Cytotherapy, 2010; 12: 792–806

Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

RAKHI PAL1, CHAITANYA GOPINATH2, NAGESH M RAO2, POULOMI BANERJEE1, VENKATESH KRISHNAMOORTHY2, NEELAM K. VENKATARAMANA3 & SATISH TOTEY3 1Stempeutics

and

Research Pvt Ltd, Manipal Hospital, Bangalore, India, 2NU Research, Padmanabhanagar, Bangalore, India, Neuroscience Allies Pvt Ltd, Indiranagar, Bangalore, India

3Advanced

Abstract Background aims. Spinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope. Pre-clinical and clinical studies have established that these cells are safe for transplantation. The dose dependency, survivability, route of administration, cell migration to injury site and effect on sensory and motor behavior in an SCI-induced paraplegic model were studied. Methods. A spinal cord contusion injury model was established in rats. Bone marrow (BM) mesenchymal stromal cells (MSC) were tagged to facilitate tracing in vivo. Two different doses (2 and 5 million cells/kg body weight) and two different routes of infusion (site of injury and lumbar puncture) were tested during and after the spinal shock period. The animals were tested post-transplantation for locomotor capacity, motor control, sensory reflex, posture and body position. Stem cell migration was observed 1 month post-transplantation in spinal cord sections. Results. The overall results demonstrated that transplantation of BM MSC significantly improved the locomotor and sensory behavior score in the experimental group compared with the sham control group, and these results were dose dependent. All the infused stem cells could be visualized at the site of injury and none was visualized at the injected site. This indicated that the cells had survived in vivo, were probably chemoattracted and had migrated to the lesion site. Conclusions. MSC transplanted with a lumbar puncture method migrate to the site of injury and are the most suitable for SCI healing. These cells demonstrate a dose-dependent effect and promote functional recovery when injected during or after the spinal shock period. Key Words: allogenic, bone marrow, lumbar puncture, mesenchymal stromal cells, spinal cord injury

Introduction Spinal cord injury (SCI) affects many people, resulting in local and distant damages to the cord followed by a range of cellular disturbances, hemostatic imbalance and ionic and neurotransmitter derangements (1). This is associated with degeneration of the spinal tracts and axons, loss of neurons and glia and abnormal secretion of myelin inhibitors such as neuritis outgrowth inhibitor (NOGO), myelin-associated glycoprotein (MAG), tenascin and chondroitin sulfate proteoglycans, which lead to demyelination in the vicinity of the lesion epicenter. In addition to this, there is a release of free radicals that leads to inflammation. These collectively lead to loss of functionality, resulting in paraplegia or quadriplegia. SCI patients also show abnormal secretion and accumulation of

neurotransmitters, resulting in excitotoxicity, which leads to severe cellular damage and cell death (2). This cellular damage and cell death further lead to secondary injury and ultimately there is formation of glial scar and cavitations that leads to regenerative failure (3). Several attempts have been made to reverse such damage. A few of them have demonstrated longdistance regeneration of adult dorsal root ganglion cells, transplanted into spinal cord pathways using a micrograft technique to escape local scarring. But axonal outgrowth was terminated or reversed upon contact with scar tissue. Therefore, stem cell therapy has emerged as an intriguing and attractive possibility in the field of transplantation medicine, especially for SCI.

Correspondence: Dr Satish Totey, President & CEO, Advanced Neuroscience Allies Pvt Ltd, No. 560, 1st Floor, 9th A Main, Indiranagar, Bangalore 560 038, India. E-mail: [email protected] (Received 29 December 2009; accepted 5 April 2010) ISSN 1465-3249 print/ISSN 1477-2566 online © 2010 Informa Healthcare DOI: 10.3109/14653249.2010.487899

Mesenchymal stem cells in Spinal cord injury Although spinal cord cells can give rise to neurons and related cells, they are incapable of producing all the cell types necessary for recovery after injury (4). Researchers envisage stem cells may (a) act as a cellular bridge, assisting axons above and below the injury site, (b) act as a new source of neurons, (c) secrete neurotropic substances (substances that have an affinity for neural tissue) that promote repair, (d) modulate the immune response after injury, (e) induce breakdown of inhibitory scars within the spinal cord and eliminate cell debris and (f) protect neurons. Several different stem cells have been tried for spinal cord regeneration in human clinical trials, including mesenchymal stromal cells (MSC) (5), human embryonic stem cells (6), olfactory ensheathing cells (7), hematopoietic stem cells (8) and umbilical cord stem cells (9), with variable degrees of recovery. However, there are still a large number of unanswered questions and stem cells have not yet been shown to be highly beneficial in clinical applications. To answer some of these questions, we examined the fate of transplanted human bone marrow (BM)-derived MSC, their migration to the site of injury, survivability and effect on sensory and motor behavior in an SCI-induced paraplegic rat model. The goal of this study was to investigate whether the transplantation of human BM MSC after contusion SCI promoted any functional outcome, as measured by behavioral tests and immunohistochemistry. Methods Isolation of human BM MSC BM MSC were isolated and expanded using a method reported previously (10). Briefly, human BM was aspirated aseptically from the iliac crest of each patient under deep sedation after obtaining informed consent. All processing of the samples was done inside a class 100 biosafety hood. The BM was diluted (1:1) with knockout Dulbecco’s modified Eagle’s medium (KODMEM; Invitrogen, Carlsbad, CA, USA). The BM was centrifuged at 1800 r.p.m. for 10 min to remove anticoagulants. The supernatant was discarded and the BM washed once with culture medium. Mononuclear cells (MNC) were isolated by layering onto a lymphoprep density gradient (1:2; Axis-Shield PoC AS, Oslo, Norway). MNC present in the buffy coat were washed again with culture medium. The mononuclear fraction, which also contained MSC, was plated onto T 75-cm2 flasks (BD Biosciences, San Jose, CA, USA) and cultured in KO-DMEM. The medium was supplemented with 10% fetal bovine serum (FBS; Hyclone, South Logan, UT), 200 mM glutamax (Invitrogen) and Pen-Strep (Invitrogen). The non-adherent cells were removed after 48 h of culture and replenished with

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fresh medium. Subsequently, the medium was replenished every 48 h. Subculturing of MSC Once the cells attained confluency, they were dissociated with 0.25% trypsin/0.53 mM ethylene diamine tetraacetic acid (EDTA; Invitrogen) and reseeded at a density of 5000 cells/cm2 into T 75-cm2 flasks. After 5–7 days in culture, the cells reached 90% confluency and were subcultured for subsequent propagation or transplantation. The cells were further up-scaled and expanded in order to provide the required number of cells for grafting into animals. Immunophenotyping Flow cytometry. Immunophenotyping of cultured BM MSC was performed as described elsewhere (10), using flow cytometry to identify the presence of specific cell-surface antigens. Briefly, BM MSC were dissociated with 0.25% trypsin–EDTA and resuspended in wash buffer at a concentration of 1 ⫻ 106 cells/mL. The wash buffer consisted of phosphate-buffered saline (PBS) supplemented with 1% (v/v) FBS and 1% (w/v) sodium azide. Cell viability was measured by flow cytometry using 7-amino actinomycin D (7AAD). Two-hundred microliters of cell suspension were incubated in the dark for 30 min at 4°C with saturating concentrations of fluorescein isothiocyanate- (FITC) or phycoerythrin- (PE) conjugated antibodies. Appropriate isotype-matched controls were used to set the instrument parameters. After incubation, cells were washed three times with wash buffer and resuspended in 0.5 mL wash buffer for analysis. Flow cytometry was performed on an LSR-II (BD Biosciences). Cells were identified by light scatter for 10 000 gated events and analyzed using FACS DIVA software (BD Biosciences). Viability was calculated using 7AAD. The following MSC markers were analyzed: CD34–PE, CD44–PE, CD45–FITC, CD73–PE, CD90–PE (BD Pharmingen, San Diego, CA, USA) and CD105–PE (R&D Systems, Minneapolis, USA). Immunocytochemistry Immunofluorescence was carried out using a method described elsewhere (5). Briefly, differentiated cells were fixed in 4% paraformaldehyde (PFA) for 1 h at 4°C. Blocking was done for 30 min at Room Temperatur (RT) with 3% Bovine Serum Albumin (BSA) to reduce non-specific binding. The cells were incubated overnight in primary antibody [glial fibrillary acidic protein (GFAP)/Olig 4 (O4) (R&D Systems), and β-III tubulin (Sigma-Aldrich)] at 4°C,

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followed by a 1-h incubation with secondary antibody. This was followed by DAPI staining in the dark for 30 min at room temperature. Finally the slides were mounted with Vectashield solution and visualized under a microscope. Images were captured using a Nikon Eclipse 90i microscope (Nikon Corporation, Japan) and Image-Pro Express software (Media Cybernetics Inc., Silver Spring, MD, USA) Differentiation potential The multipotent capacity of the cells was determined by their differentiation potential into adipocytes and osteoblasts (10). Briefly, osteoblast differentiation was induced by culturing human BM MSC in KO-DMEM supplemented with 10% FBS (Hyclone), 200 mM glutamax (Invitrogen), 10–8 M dexamethasone (SigmaAldrich), 30 μgm/mL ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich) for 3 weeks. Fresh medium was replenished every 3 days. Calcium accumulation was assessed by Von Kossa staining. To induce adipogenic differentiation, human BM MSC were cultured for 21 days in KO-DMEM supplemented with 10% FBS, 200 mM glutamax, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 1 μg/mL insulin and 100 μM indomethacin (all Sigma-Aldrich). Inducing factors were added to the replenished medium every 3 days. Tagging/tracking of cells For in vivo tracing and identification of the cells after migration, BM MSC were labeled with a PKH26-CL kit (Sigma-Aldrich). Briefly, the cells were harvested using 0.25% trypsin–EDTA (Invitrogen) and the cell number calculated using a standard hemocytometer. The cells were washed with serum-free medium and suspended in diluent ‘C’ as provided in the kit. The cell suspension was then stained with diluted PKH26 dye for 5 min, neutralized and centrifuged at 1500 r.p.m. for 5 min. Animal experiments Selection and screening of animals. Seventy-two animals were used in the study. Appropriate approval was obtained from the Institutional Animal Ethics Committee (IAEC) of the NU Trust (CA 6, 15th main, 11th cross, padmanabhanagar, Bangalore-560070, Karnataka, India) and performed in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and recommendations. The animals were procured from the Central Animal Facility (CAF) of the Indian Institute of Science (IISc, Bangalore, India). All animal experiments were carried out at the animal research facility at NU Research

Figure 1. The study design for the animal experiment.

(Bangalore, India). The design of the animal experiment is given in Figure 1. Adult male Wistar rats, with an average age of 4 weeks and body weight of 150–200 g, were selected for the study. The rats were housed individually in plastic cages, maintained on a 12-h light/dark cycle, and had free access to food and water. The animals were screened on a grid walk and Basso–Beattie–Bresnahan (BBB) locomotor scale scoring was performed. Only those animals that recorded no foot slips and scored 21 on the BBB were selected for study. SCI model Surgical procedure. All surgical procedures were performed under deep anesthesia induced by intramuscular administration of 80 mg/kg body weight ketamine (Aneket 500 mg; Neon Laboratories Pvt Ltd, Thane, India) and xylazine (20 mg; Indian Immunologicals Ltd, Guntur, India). The anesthetic plane was determined by withdrawal to foot/tail pinch. Each animal received a pre-operative dose of 2.5 mL ringer lactate solution subcutaneously, and gentamycin (Genticyn 20 mg; Nicholas Piramal India Ltd, Mumbai, Maharashtra, India) and bupivucaine (Anawin 0.25%; Neon Laboratories Ltd, Mumbai, India) intramuscularly. The body temperature was monitored during surgery and maintained using an electrical heating pad.The eyes were kept moist throughout the surgery using saline drops. The model The rats were placed in dorsal recumbence, and the surgical sites shaved and cleaned with betadine. A 150-Kdyne spinal cord contusion injury was delivered using a PSI-Infinite Horizon impactor as described previously (11). Briefly, following an

Mesenchymal stem cells in Spinal cord injury incision through skin, subcutis and muscle, the paraspinal muscles were retracted laterally and a dorsal laminectomy at T8–T9 was performed (identified by a vein crossing the processus spinosus of T4 and T5). Next, the vertebral column was stabilized with forceps on the impactor surgical surface; the tip of the impactor adjusted to the site of laminectomy and, with an intact dura mater, the spinal cord was impacted with 150 Kdyne force, 0 s dwelling time and 1 mm/s velocity, as delivered through the Infinite Horizon impactor software. The actual force impacted on the cord, velocity and dwell time were recorded by the software. The surgical wound was then closed in layers using a 12-sized suture needle (Surgeon, Quality Needles Pvt Ltd, Uttar Pradesh, India) and 4.0 sutures (Centenial Surgical Suture Ltd, Thane, India). Post-operative care Post-operative care of the animals was carried out following contusion injury. This included manual expression of the bladder twice a day for the first 7 days post-injury, followed by once daily for 3–4 days or until the animal regained autonomous bladder activity, and daily administration of gentamycin and cefazolin (Reflin 250 mg; Ranbaxy Laboratories, Mumbai, India) for 7 days postcontusion. Grafting of human adult BM MS Intrathecal delivery of cells: lumbar puncture. The rats were anesthetized with a ketamine/xylazine cocktail as mentioned above, placed in a dorsal recumbent position and the surgical sites shaved and swabbed aseptically. A skin-deep longitudinal incision was made over the L3–L5 processes. Each animal was placed on the edge of a platform and its tail held firmly down, perpendicular to the plane of its body. This helps to increase the space between the vertebrae and thus locate it. The lumbar space was identified and a neonatal 26-G ½-inch needle Becton Dickenson (BD) was introduced into the lumbar space between L3 and L4 or L4 and L5 and held in position. The location of the needle into the lumbar space was confirmed by three confirmatory signs during the procedure: (a) a feeling of ‘give’, (b) tail flick and (c) the presence of cerebrospinal fluid (CSF) in the needle hub. A Hamilton syringe was secured onto the needle and held firmly in position. Tagged BM MSC were injected into the CSF using a 500-μL Hamilton syringe, which delivers at the rate of 7.26 μL/rotation over a period of 1 min. The breathing of the animal was closely monitored during the transplantation process. The needle was kept in position for 5 min

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to prevent any reflux of cell suspension and gently removed afterwards. Pressure was applied to the lumbar puncture (LP) site for 2 min to prevent any leakage from the needle tract. Thereafter, the skin was sutured aseptically and the animal returned to its cage and provided with 2 mL ringer lactate solution, antibiotics and analgesics. The body temperature was maintained till the animal regained consciousness and post-operative care was initiated for 7 days. Delivery of MSC into the parenchyma: at the lesion site For grafting of cells into the site of injury, a glass micropipette with a tip diameter of approximately 100 μm was fixed onto a 26½-gauge needle. The bore of the micropipette was checked for any blockage before transplantation. This was fixed to a 500-μL Hamilton syringe, which delivers at the rate of 7.26 μL/rotation. Each rat was anesthetized with a ketamine/xylazine cocktail, placed in a dorsal recumbent position and the, surgical sites shaved and swabbed aseptically. An incision was made at the site of injury over the T8–T9 processes and the underlying sutures located/ removed. The injury site was cleared of any additional tissue that may have grown as a part of the healing process. The injury site was located and cleared such that the lesion was clearly visible. The cells were delivered over a period of 2 min to regions rostral and caudal to the lesion site. Glass capillaries were used to enhance graft survival and reduce the local glial response compared with transplantation using metal cannulas (12), although the risk of breaking within the tissue was quite high. Assessment of sensory-motor functions: behavioral tests Prior to all behavioral testing, the bladder of the all the animals was expressed and food and water provided ad libitum. Open-field test The BBB locomotor scale was used to assess locomotor recovery in this animal model. Observers blinded to the study performed the test. Before each session, for each animal the bladder was expressed to prevent any hind limb-related activity due to spontaneous bladder contractions. The open-field locomotion score (BBB score) was designed to measure the recovery of hind limb movements after SCI in rats during free, open-field locomotion. For this purpose, the rats were placed in an open field (80 ⫻ 130 ⫻ 30 cm) with a non-slippery floor. In each testing session, the animals were observed individually for

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4 min. Hind limb locomotion was then scored from 0 to 21 points. A score of 0 defines no movement of the hind limb, and the maximum of 21 defines normal locomotion as observed in unlesioned rats. Points were distributed according to criteria such as joint movement, weight support, fore limb–hind limb co-ordination and tail position. Grid walk Deficits in descending motor control were examined with the grid walk task. Because of the spacing of the grid bars, this task requires a controlled placement of the hind limbs that depends on supra-spinal input in part through the cerebrospinal tract (CST). Animals were trained for 3 days before baseline evaluation. For analysis of the grid walk performance, the rats were allowed to cross the grid twice. The total number of footfalls was calculated. A footfall was defined as a drop of the foot below the plane of the grid. Animals that could not support their weight on their hind limbs or froze on the grid would make errors with every step and were thus assigned 100% footfalls.

started with an incline of 30° and both directions were tested, to detect any asymmetries between limbs. The highest angle at which a rat was able to maintain a horizontal position for 10 s without slipping was recorded. If the hind limb of an animal slipped or a rat walked during its trial, this indicated a negative trial and it was repeated at the next lowest angle. With every successful attempt, the inclined plane was increased by 10°. With an unsuccessful attempt, it was repeated at the same angle and after three unsuccessful attempts the angle was reduced by 5°. Perfusion of animals Four weeks post-transplantation, all the animals in both groups were killed. The animals were anesthetized with a ketamine/xylazine cocktail, followed by transcardial perfusion with 0.9% heparinized saline and 4% paraformaldehyde at room temperature; 0.9% heparinized saline 200 mL was infused until the circulation was clear of traces of blood. Later on, animals were infused with 100 mL 4% PFA until stiffness set in completely. Processing of tissue

Plantar test The Plantar test (Hargreaves’ method) discerns a peripherally mediated response to thermal stimulation caused by external sources in an unrestrained rat. The system consists of movable infrared source, a glass pane onto which the rat enclosure is located and a controller unit. A rat was placed in one of three compartments. After an acclimation period, the infrared source was placed under the glass floor and positioned directly beneath the hind paw. A trial was commenced by depressing a key that turned on the infrared source and started a digital solid state timer. When the rat felt pain it withdrew its paw. The withdrawal of the paw caused a sudden drop in the reflected radiation, which switched off the infrared source and stopped the reaction time counter. The withdrawal latency was calculated to the nearest 0.1 s. Three such readings were taken for each hind paw (right and left) at an interval of 5 min each. Inclined plane The inclined plane consists of a horizontally grooved rubber surface adjusted in 5° increments from 0° to 90° using adjustable protractor hinges. A foam pad was placed at the base as a safety measure for the animals. Each animal was placed horizontally on the mat surface facing either to the right or left, until it was restrained properly and calmly without griping the edges of the plane with its tail. The test was

After perfusion, the spinal column was removed and preserved in 4% PFA for 2–3 days. The spinal cord was dissected out from the vertebral column and placed in 4% PFA for 24 h, followed by 30% sucrose at 4°C till the tissue sunk. Histopathology Cryosection. Histologic evaluations were performed on 10- and 20-μm cryosections made from the spinal cord tissue and adhered on to albumin pre-treated slides. This procedure was done using a Shandon Thermo Scientific cryostat, USA. Preparation of slides for cryosections Fresh egg albumin was diluted 1:1 with Reverse Osmosis (RO) water and mixed well. Glass slides (blue star) were cleaned with soap water and allowed to dry overnight. One drop of diluted egg albumin was smeared uniformly and evenly on to each slide and left to air dry overnight at room temperature. Preparation of tissue for cryosections The spinal cord tissue was taken out of the 30% sucrose solution and dried with Kim wipes. The tissue was trimmed according to the areas of interest, namely the site of injury and LP site. A rectangular cryoblock was prepared with Shandon cryomatrix (Thermo Scientific), placed on

Mesenchymal stem cells in Spinal cord injury a cryobar (temperature –80°C) inside a cryostat chamber (ambient temperature –20°C) and allowed to solidify. The tissue was placed in the cryoblock and covered with more cryomatrix to embed the tissue in the block. It was placed on the cryobar for 30 min to solidify. The block was then placed in the cryotome tissue holder and set to either 10 microns or 20 microns and serial sections were taken on the glass slides. Cell tracking The sections were screened under a fluorescent microscope (Nikon 90i) Tetramethyl Rhodamine Iso-Thiocyanate (TRITC) channel to visualize the presence of BM MSC that had been pre-stained with the PKH26 as mentioned above. Statistical analysis Descriptive statistical analysis was carried out. Continuous measurements are presented as mean ⫾ SD. Significance was assessed at the 5% level. A Student’s t-test (two-tailed, independent) and analysis of variance was used to find the significance of parameters between two/three groups. A 2 ⫻ 2 ⫻ 3 repeated measure ANOVA was performed to find the significance of the interaction effect and the main effect in a repeated-measures design. Results Animal care and recovery post-SCI The mean body weight of all rats during the course of the study did not differ significantly (250 ⫾ 25 g). There was some loss of body weight in the first 5 days post-contusion that stabilized with time. Bladder expression was continued for 2 weeks

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post-injury and antibiotics were given to animals that showed hematuria or other signs of urinary tract infection. Viability and characterization of BM MSC BM MSC were characterized as described in the Methods. These cells were tested for CD34, CD44, CD45, CD73, CD90, CD105 and HLA-DR. The cells were negative for CD34, CD45 and HLA-DR and brightly positive for CD73, CD90 and CD105, representing a true mesenchymal phenotype. The viability of these cells was more than 90%, as demonstrated by 7AAD staining (Figure 2).

Grafted BM MSC improved locomotion BM MSC were tagged with PKH26 after harvesting. These cells were transplanted into the rats by two different methods, the first at the site of injury and the second via LP. Two different doses were used in this study, 2 million cells/kg body weight and 5 million cells/kg body weight. Cells were injected either on the third day post-injury, during the spinal shock period (group I), or on the 14th day post-injury, during the post-spinal shock period (group II). The overall results demonstrated that transplantation of BM MSC improved locomotor and sensory behavioral scores in the experimental group of rats. The sham group showed some improvement but significantly less than the experimental group. The locomotor capacity of the animals was tested with the BBB score, deficits in descending motor control were examined by grid walk, sensory reflex with the plantar test and ability to maintain body position with appropriate posture using the inclined plane.

Figure 2. The immunophenotype of the BM MSC used for transplantation in contused rats. The cells were CD34– CD73⫹ CD90⫹ CD44⫹ CD105⫹ CD166⫹.

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Figure 3. BBB scores of animals in the different groups during the course of the experiment. The baseline depicts the scores of normal healthy animals before injury. Post-contusion indicates the score 48 h after the injury. Days 7, 14, 21, and 30 are days posttransplantation. (A) BBB scores for group I LP. (B) BBB scores for group II LP. (C) BBB scores for group I site. (D) BBB scores for group II site.

BBB score The BBB locomotor score was determined in animals before SCI and at 1, 7, 14, 21 and 30 days post-transplantation. All rats scored the maximum of 21 before SCI. After 24 h of spinal cord contusion injury, all rats were completely paraplegic, with a BBB score of 0 (Figure 3). The control, salinetreated rats in both groups gradually regained some movement with time (11.67 ⫾ 0.82 at day 30) but not to the level of the cell-transplanted group. In the LP-treated group I, the BBB scores were significantly higher on days 7, 14, 21 and 30 than those seen in sham control animals (P ⬍ 0.001). However, there was no significant difference between 2 million cells (12.1 ⫾ 0.41) and 5 million cells (13.0 ⫾ 0.8) and occasional to frequent forelimb to hind limb co-ordination (Figure 3A) was seen. Similarly, in group II both 2 million and 5 million cells showed significantly higher BBB scores than the sham-treated group, although the 2 million group scored 15.33 ⫾ 0.82 (indicating no toe clearance, parallel paw position at initial contact) whereas the 5 million/kg body weight transplantation group scored 16.17 ⫾ 0.98 (indicating frequent toe clearance, parallel paw position at initial contact and rotated at lift off) (Figure 3B). In particular, LP group II animals scored more on the BBB scale than the other groups, with the dose of 5 million cells/kg body weight scoring the highest (Figure 3B). While performing the open field test, the LP group II animals with a dose of 5 million

cells/kg body weight scored the best amongst all the groups In group I, where BM MSC was injected at the site of injury (Figure 3C), the open-field testing scores were significantly higher than the sham control group (P ⬍ 0.001) and the BBB scale one point higher compared with the LP group, indicating frequent to consistent hind limb–forelimb co-ordination. The site-treated group II scored significantly higher than the sham control group and was comparable to site group I. The 2 million cells/kg body weight group scored 13.67 ⫾ 0.52 and the 5 million cells/kg body weight group 13.33 ⫾ 2.34 (Figure 3D) Grid walk The ability to control and place the hind limbs precisely was tested on a horizontal, ladderlike grid, as described in the Methods. The rats were acclimatized to the grid at the start of the experiment. Only those rats able to cross the grid with one foot slip or less were chosen for the study. Post-SCI, rats of all groups were able to cross the grid but without accurate placement of the hind paws, resulting in a high number of foot slips. Rats in group I LP dose of 5 million cells/kg body weight (Figure 4A) showed significantly fewer numbers of foot slips from day 7, reaching 18.50 ⫾ 1.76 on day 30, compared with the sham group and the dose of 2 million cells/kg body weight in the same group. In group II LP dose of

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Figure 4. Grid walk scores of animals in the different groups during the course of the experiment. The baseline depicts the scores of normal healthy animals before injury. Post-contusion indicates the score 48 h after the injury. Days 7, 14, 21, and 30 are days posttransplantation. (A) Grid walk scores for group I LP. (B) Grid walk scores for group II LP. (C) Grid walk scores for group I site. (D) Grid walk scores for group II site.

5 million cells/kg body weight, 14.67 ⫾ 3.67 foot slips were recorded, which was significantly lower than the dose of 2 million cells/kg body weight and sham-treated rats of the same group (Figure 4B). However, rats transplanted at the site of injury in groups I and II at both doses did not show any significant reduction in foot slips compared with the sham group (Figure 4C, D). The LP group II with a dose of 5 million cells/kg body weight recorded the least number of foot slips (Figure 4B) amongst all the groups, and this was significant (P ⬍ 0.001).

was not observed in the LP group I. In a few animals it was not possible to take readings between 24 h and 7 days post-contusion because they were not able to place their hind limbs plantar to the Infra-red (IR) source. There was no significant difference between the different routes of injection or timing or dose in the plantar test. The plantar test, which is a peripherally mediated response to thermal stimulation, demonstrated a similar response time between the spinal shock (group I) and post-spinal shock groups (group II). Inclined plane

Plantar test The plantar test was used to identify any changes in sensitivity. Twenty-four hours post-SCI, the rats did not show any significant increase in latency of hind limb withdrawal (Figure 5A–D). In control rats, the reaction time did not significantly shorten during the evaluation period. The rats from group II demonstrated a significantly shortened response time to the thermal stimulus compared with the controls (p ⬍ 0.001). In site group I, hypersensitivity (less than baseline values) was noted at day 14 for doses of both 2 and 5 million cells/kg body weight groups. Five million cells/kg body weight groups recovered to pre-contusion levels but the hypersensitivity was persistent in the 2 million groups. This

This test was used as an index of hind limb strength. The maximum angle at which a rat was able to maintain its position was defined as the inclined plane score. This was 70° in all animals pre-operatively. Twenty-four hours post-contusion, the animals could maintain balance at an angle of only 30°. The rats in the transplanted groups showed significantly higher values (69.58 ⫾ 1.02° in LP group I 5 million cells/kg body weight and 66.25 ⫾ 2.09° in LP group II 2 million cells/kg body weight) compared with the sham control group (55.00 ⫾ 3.54°). The LP groups recorded better scores compared with the site groups of animals (Figure 6A, B). Overall, LP group I 5 million cells/kg body weight (69.58 ⫾ 1.02°) animals scored the maximum,

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Figure 5. Plantar test scores of animals in the different groups during the course of the experiment. The baseline depicts the scores of normal healthy animals before injury. Post-contusion indicates the score 48 h after the injury. Days 7, 14, 21, and 30 are days posttransplantation. (A) Plantar test scores for group I LP. (B) Plantar test scores for group II LP. (C) Plantar test scores for group I site. (D) Plantar test scores for group II site.

almost recovering to pre-contusion levels (70°) 30 days after transplantation, and this was statistically significant (P ⬍ 0.001) In the inclined plane test, which primarily examines an animal’s capacity to balance itself and co-ordinate such an act, group I scored better than group II animals. However, there was no significant difference in recovery between group I (spinal shock) and group II (after spinal shock) animals and between 2 million and 5 million doses. Survival and migration of injected human MSC LP. In order to check for homing and migration of cells from the site of injection to the site of injury, serial spinal cord cryosections were cut at the site of injury and at the site of LP. As demonstrated in Figure 7A, B and Figure 7D, E, cells could only be visualized at the site of injury and no cells were present at the site of injection (lumbar region; Figure 7C, F). This indicated that the cells were probably chemo-attracted and had migrated to the lesion site. The cells were arranged around the lesion region or in the interconnecting tissue formed in the lesion cavity because of the injury created. A series of sections demonstrated that large numbers of BM MSC survived for at least 30 days post-transplantation.

The distribution and migration pattern of the transplanted BM MSC in the transplanted groups did not show any apparent differences. Site of injury Cells injected at the site of injury were observed to concentrate and arrange themselves bordering on the lesion cavity (Figure 8B–E) in both groups I and II. Tagged cells could not be identified in other sections of the spinal cord. Sham-operated controls did not demonstrate any tagged cells around the lesion cavity (Figure 8F, G). Transplanted BMSC do not transdifferentiate into neuroglial lineage in vitro Cryosections of spinal cord were stained for neuroglial transdifferentiation markers β-III tubulin, O4 and GFAP. The sections were found to be negative for β-III tubulin, O4 and GFAP, indicating that these cells do not transdifferentiate in vivo (Figure 9). Route of administration Two routes of injection were employed: cells were either transplanted into the animals directly at the

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Figure 6. Inclined plane scores of animals in the different groups during the course of the experiment. The baseline depicts the scores of normal healthy animals before injury. Post-contusion indicates the score 48 h after the injury. Days 7, 14, 21, and 30 are days posttransplantation. (A) Inclined plane scores for group I LP. (B) Inclined plane scores for group II LP. (C) Inclined plane scores for group I site. (D) Inclined plane scores for group II site.

site of injury or by LP technique. Recovery of the animals in both groups was compared in order to understand which route of injection was more effective. Both sets of animals showed recovery, but those animals injected via the LP route demonstrated better recovery than the site group in all the behavioral tests. The recovery was significantly higher for the LP group of animals injected 14 days post-injury. The site of injury injection was more effective in group I but was not significantly different from the LP group. Clinically LP is a much safer and approachable route for cell delivery compared with the site of injury. Extrapolating to human trials, site of injury manipulation certainly involves a greater risk of loss of residual function as well as further damage to the cord by the tract caused as a result of injecting cells into the cord. Hence we concluded that LP was the safest and most effective route of administration of cells for SCI.

Timing of transplantation Cells were injected either within 3 days of injury (spinal shock group) or 14 days post-injury (postspinal shock group). No difference was noted in the

recovery pattern when stem cells were injected during spinal shock or after spinal shock via either route of transplantation. Discussion This study was an attempt to understand comprehensively the regenerative potential of BM MSC in SCI. On a therapeutic platform, it is essential to answer a few questions before performing any clinical pilot studies. It is important to know first the effective dose of cells for transplantation, secondly which route of infusion is beneficial to obtain the desired effect of regeneration of the spinal cord, and thirdly what is an appropriate time for transplantation post-SCI. In our pre-clinical study we used two therapeutic doses, 2 million cells/kg body weight and 5 million cells/kg body weight. We divided the experimental animals into three groups for transplantation, one transplanted during the spinal shock period, the second after spinal shock and the third treated as sham. The behavioral parameters were studied over 30 days after stem cell transplantation of all animals. We observed that higher doses of stem cells (5 million cells/kg body weight) showed significantly better improvement in locomotory functions than the lower dose (2 million cells per kg body weight). Recovery

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Figure 7. Cross-section of the SCI contusion site of LP group I. The outline in white indicates the spared tissue at the injury site and BM MSC have homed into this tissue (white arrow). The MSC are labeled with PKH26 (red) and are found within the lesion site. The section has been counterstained with DAPI (blue). Magnification ⫻10. (A) Site of injury LP group I 2 million. (B) Site of injury LP group I 5 million. (C) The LP region in LP group I. (D) LP group II 2 million. (E) LP group II 5 million. (F) LP region of LP group II animals.

was found to be higher in the 5 million/kg body weight LP group of animals compared with the 5 million/kg body weight site group of animals. This was probably because of the minimal manipulation of tissue using the LP technique in contrast to the site of injection, where the needle had to be introduced into the tissue for cell delivery. After cell transplantation, the cells migrated to the lesion site, survived for more than 30 days and facilitated recovery. Although there is evidence in the literature that suggests these cells have the capability to transdifferentiate into neural cells or progenitors (13), their mechanism of action is to date still highly controversial. Moreover, in our experiments most of these cells were negative for astroglial markers, suggesting that transplanted BM MSC did not transdifferentiate into lineage-specific cells. It is possible that these cells need to reside in the lesion area for a longer duration (more than 30 days) before they can express any properties of differentiation. So far, the probable mechanism of action of BM MSC may be a paracrine effect of the soluble trophic factors that they secrete around the injury region, rather than transdifferentiating into neuronal lineages. These results are in concurrence with other studies where it has been suggested that BM MSC secrete bioactive molecules and mediate the proliferation and homing of

endogenous stem cells from their niche to the injured location (9,14–16). In this study, human BM MSC were injected into adult male Wistar rats. During the course of the study (before and after transplantation) no immunosuppressant was administered. At the end of 30 days post-transplantation of BM MSC, we were able to detect human BM MSC at the site of injury in both the LP and site groups. This indicated that the MSC had survived and migrated to the injury site and had not elicited any immunogenic reaction in the host tissue. It also suggested that BM MSC are hypo-immunogenic in nature and are well tolerated by rats post-transplantation without immunosuppression. This could be explained by the absence of HLA-DR (as demonstrated in this study) and low expression of co-stimulatory molecules, leading to inhibition of naive and memory T-cell responses, as well as B-lymphocyte attenuation for antibody production as demonstrated by others (17). The results indicate that BM MSC do not evoke any graft versus host immune reaction and have the capacity to escape the immune system and yet be effective in wound healing. Earlier studies have demonstrated that BM MSC are hypo-immunogenic and evade allo-rejection (17–19). This study reconfirms a very clinically relevant observation, indicating that allogenic BM MSC may be transplanted into

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Figure 8. Section of the contusion site for SCI rats in site group II. The outline in white indicates the cavity created as a result of the injury. MSC are labeled with PKH26 (red) and are found around the lesion site (white arrow). The sections have been counterstained with DAPI (blue). Magnification ⫻10. (A) MSC tagged with PKH26 in vitro (red) counterstained with DAPI (nuclei blue). (B) Site group II 2 million cells/kg body weight. (C) Site group II 5 million cells/kg body weight. (D) Site group I 2 million cells/kg body weight. (E) Site group I 5 million cells/kg body weight.

Figure 9. Transverse section of the SCI contusion site of the transplanted group of animals. The interconnecting tissue at the centre of the lesion was stained with DAPI (blue) (A) and the BM MSC at the lesion site was stained with PKH26 (red). The transplanted cells do not express β-III tubulin or GFAP. Magnification ⫻10.

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humans without using any immunosuppressant and would not elicit an immune response in the host tissue, unlike other studies (14,20,21). This study did not reveal any difference in the recovery pattern when BM MSC were injected during the spinal shock (3 days of injury) or after spinal shock (14 days after injury) periods. This provides evidence that, even after 72 h of injury, the environment of the lesion site is not hostile. Stem cells not only survived but also exerted their desired effect. It is known that inflammation plays an important role during an insult to the nervous system. There is a well-know acute phase followed by a prolonged inflammatory phase characterized by the release of inflammatory cytokines, leukocytes, monocytes and macrophages at the site of injury, but these seem to subside within 72 h, allowing the BM MSC to lodge at the injury site, remain viable and promote regeneration. In an earlier report, stem cells were injected immediately after injury, overlapping with the immediate acute phase (primary events) of the injury (22). This resulted in very low or no improvement of motor function, possibly because the cells had been subjected to the inflammatory response as a result of the injury, resulting in cell death. Probably by 72 h the level of inflammation and production of macrophages, leukocytes and free radicals has reduced to a significantly low level, allowing the BM MSC to prevent any further damage. We have also demonstrated that, after 14 days of injury when cells were injected, there was a similar pattern of recovery as noted in the earlier group. This implies that, once the acute phase of destruction and the physiologic responses to it are over, stem cells can be injected to initiate recovery. It has been demonstrated that stem cells transplanted 1 day post-contusion injury by LP showed better cell survival, migration and cell grafting at the lesion site (20,23). However, functional recovery was not studied and all the animals had been administered with immunosuppressants (20,24). In an another study, stem cells were injected immediately and 7 days post-contusion and better recovery was reported in the later group (22). Combining these data with those obtained from other studies (25,26), BM MSC may be injected as early as 3 days post-injury to remain viable and effective at the lesion site. Also, BM MSC are equally effective when injected 14 days after the lesion, during the post-spinal shock period. This implies that BM MSC may be effective in acute and subacute cases of SCI. Therapeutically, in humans this translates to the fact that BM MSC may be injected into a patient during the later phase of the spinal shock period at the earliest. This also implies that there is ample time for primary procedures, such as spinal stabilization,

after which cell therapy intervention can be initiated. This may limit any further injury from occurring, but this needs further substantiation. The route of injection is another important factor that needs to be taken into account when considering cell therapy for SCI patients. The routes of administration that may be considered are intravenous, at the site of injury, intraventricular and LP. In this study, intravenous (27,28) intra-ventricular (24) and intrastriatal have not been considered. While considering any route of administration, it is essential not to produce any further damage to the cord and simultaneously be able to produce maximum benefit from the transplanted cells. Injection at the site of injury assures the delivery of cells into the lesion site. It is important to note that this requires a major neurosurgical manipulation with multiple risks involved. First, the risk from anesthesia is quite high in SCI patients; secondly, the patients must undergo laminectomy, which will further destabilize the already compromised state of the vertebral column, leading to further spinal deformities in the future. Most SCI patients have altered blood flow, inflammation and distortions in the spine as a result of injury, or a decompression surgery conducted earlier and implants present to stabilize the spine. These will make the spinal cord even more difficult for the neurosurgeon to access. For transplantation of cells into the lesion site, it is also essential to remove the dura mater from over the area. This makes the region more prone to leakage of CSF and increases the incidence of infection, leading to more inflammation and scarring of already damaged neural tissue. A leak-proof closure of the dura is essential to prevent cells from flowing out of the transplantation site. Finally, translating to clinical trials, if multiple injections/doses are required it would be technically and practically impossible to repeatedly transplant at the lesion site. All these facts together make transplantation of cells at the site a difficult proposition to translate into clinical trials and hence clinical therapy. Intravenous injection of cells is another viable option. But, as demonstrated earlier (29), the cells tend to lodge in other major organs (such as lungs and liver) before reaching the lesion site. Particularly because SCI can occur as a result of road traffic accidents, and patients suffer from multiple injuries, the cells tend to migrate to all the injured locations. In such a scenario the required number of cells may not reach the lesion site. Also, as evidenced in literature (30–32), the blood–brain/spinal cord barrier has increased permeability during the acute phase of SCI. Hence cells would reach the injured spinal cord only if there was a breach in the blood–spinal cord barrier/blood–brain barrier. This implies that the cells would cross over the lesion region only when injected during this time window. Intraventricular injection also involves similar risks as site of injury injection

Mesenchymal stem cells in Spinal cord injury and hence would not be a viable option. LP is a routinely practiced minimally invasive procedure in clinics. In this procedure, a needle is introduced into the lumbar region of the spine (which houses the terminal nerve fibers and none of the major spinal tracts). Because of the nature of the procedure, it does not require anesthesia and involves minimum manipulation and damage to the spinal cord. The most commonly reported complication after LP is headache, which can be abolished if minimal CSF is withdrawn during the procedure. In our study, we observed that the LP group of animals improved more than the site of injury group of animals. The reasons mentioned earlier may be attributed to this observation. During the course of the study it was also observed that the site of injection in animals was more fragile and prone to infections after repeated exposure of the spinal cord. This was not observed in the LP group of animals. Also in the LP group of animals, cells migrated to the site of injury from the LP site, as evidenced by the histopathogic data. The minimal manipulation of the spinal cord probably facilitated the recovery of the animals and yielded better results compared with the site of injury group. These results are similar to earlier reports that have demonstrated migration of stem cells to the injured region when injected by LP, and functional recovery (22–24). To conclude, although recovery was slightly better in the 5 million cells/kg body weight group compared with 2 million cells/kg body weight group, it was found that recovery may not be a dose-dependent phenomenon. Perhaps the most important factor for healing was the number of cells that survived at the lesion site after transplantation. This study suggests that injection of cells can be carried out during either the spinal shock period or the post-spinal shock period to yield comparative results. The LP route of stem cell infusion is convenient, safe and more efficacious than site of injury transplantation and allows multiple infusions without any risk of infection and surgical interventions. Acknowledgments We would like to acknowledge the staff at the Laboratory Medicine Department of BGS Global Hospital for support with the cryostat, the staff at the Central Animal Facility at the Indian Institute of Science, Bangalore, for providing SPF animals, and Dr K. P Suresh for assistance with the bio-statistical analysis. Declaration of interest: The authors report no conflict of interest and are responsible for the content and writing of the paper.

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