Acta Neurochir (2014) 156:1409–1418 DOI 10.1007/s00701-014-2089-6
EXPERIMENTAL RESEARCH - BRAIN INJURY
Transplantation of neurotrophin-3-expressing bone mesenchymal stem cells improves recovery in a rat model of spinal cord injury Ling-Jie Wang & Rui-Ping Zhang & Jian-Ding Li
Received: 10 January 2014 / Accepted: 27 March 2014 / Published online: 18 April 2014 # Springer-Verlag Wien 2014
Abstract Background This study aimed to investigate the therapeutic effects of transplanting neutrophin-3 (NT-3)-expressing bone marrow-derived mesenchymal stem cells (BMSCs) in a rat model of spinal cord injury (SCI). Methods Forty-eight adult female Sprague–Dawley rats were randomly assigned to three groups: the control, BMSC, and NT-3-BMSC groups. BMSCs were infected with NT-3DsRed or DsRed lentivirus and injected into the cerebrospinal fluid (CSF) via lumbar puncture (LP) 7 days after SCI in the NT-3-BMSC and BMSC groups, respectively. The hind-limb motor function of all rats was recorded using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale on days 1, 3, 7, 14, 21, 28, and 35 after transplantation. Haematoxylineosin (HE) staining, immunofluorescence labelling, and western blotting were performed at the final time point. Results Expressions of NT-3, brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) proteins increased significantly in the NT-3-BMSC group, and hind-limb locomotor functions improved significantly in the NT-3-BMSC group compared with the other two groups. The cystic cavity area was smallest in the NT-3-BMSC group. In the NT-3-BMSC group, neurofilament 200 (NF200) and glial fibrillary acidic protein (GFAP) expression levels around the lesions were significantly increased and decreased, respectively.
L.DsRed Express2) were constructed and then packaged into lentiviruses with three helper plasmids (pLV/ helper-SL3, pLV/helper-SL4, and pLV/helper-SL5) in 293 T cells by Cyagen Biosciences Inc. (Suzhou, China). The dilution method of plate counting test revealed that the titres of the NTF3-DsRed lentivirus and DsRed lentivirus ranged from 1– 1.8×108 TU/mL and 3.5−3.8×108 TU/mL, respectively. The BMSCs (2×105) in passage 3 were infected with NTF3DsRed lentivirus or DsRed lentivirus following the standard procedures of infection. NTF3-DsRed BMSCs and DsRed BMSCs were harvested, and NTF3-DsRed gene and DsRed gene expressions were detected using a fluorescence microscope (IX71, Olympus, Tokyo, Japan). NTF3 protein was detected by western blotting.
BMSC isolation BMSCs were isolated from the tibias and femurs of 8-weekold female Sprague–Dawley rats (Animal Centre of Chinese People’s Army Military Medical and Scientific Academy, Beijing, China, SCXK2007-004). Donor rats were killed with intraperitoneal injections of an overdose of pentobarbital sodium, and the tibia and femur were dissected. After scraping off all the attached muscles and cutting of both ends of the bones, the marrow was obtained by flushing the bone marrow cavity with 5 mL Dulbecco’s modified essential medium (DMEM; Hyclone, Logan, UT, USA) using a 25-gauge needle. The collected bone marrow was made into a single-cell suspension by triturating with a pipette, and then filtered through a 100-μm mesh nylon cell strainer. After the suspension was centrifuged at 1,500 rpm for 10 min at room temperature, the supernatant was removed. The obtained marrow cells were plated at a cell density of 109/L in 25-cm2 culture flasks in DMEM containing 10 % foetal bovine serum
Preparation of the SCI model Adult female Sprague Dawley rats (8–10 weeks old, weighing 180–240 g) were anaesthetised with an intraperitoneal injection of pentobarbital sodium (50 mg/kg), and laminectomy was performed microscopically on the T7−8 vertebrae. The SCI model was generated using a weight-drop device [6]. A metal rod with a weight of 25 g and diameter of 2 mm was dropped from a height of 3 cm onto the exposed spinal cord to induce a contusion lesion. The twitching hind limbs of the rats completely relaxed, indicating that the SCI model was successful. Then, the muscles and skin were tightly sutured in layers. After surgery, 2×105 U penicillin was given via intramuscular injection every day to prevent infection, and manual emptying of the bladder was performed twice a day until recovery of the micturition reflex. Rats without hind-limb paralysis were excluded.
Acta Neurochir (2014) 156:1409–1418
1411
Group allocation and cell transplantation
Immunofluorescence labelling
A week after contusion, 48 rats were randomly assigned to the following three groups (n=16/group): the control, BMSC, and NT-3-BMSC groups. The rats were re-anaesthetised, and cell transplantation via lumbar puncture (LP) was performed as described previously [1]. In brief, at the L4–L5 intervertebral space, a LP needle (25 gauge) was advanced into the subarachnoid space, and 1×106 cells diluted in 40 μL culture medium were injected into the cerebrospinal fluid (CSF) with a microsyringe. In the control group, the rats received 40 μL culture media without any media or cells via the CSF. For the BMSC and NT-3-BMSC groups, the rats received BMSCs and NT-3-BMSCs, respectively. After cell transplantation, each rat was kept on a 30° slope in the head-down position for 30 min [31].
The 5-μm-thick longitudinal frozen sections were fixed in cold acetone for 30 min. The sections were treated with 0.3 % Triton X-100 in phosphate-buffered saline (PBS) for 30 min at room temperature and immunoblocked with 10 % goat serum in PBS for 1 h at room temperature. Then, they were incubated overnight at 4 °C with the primary antibodies: neurofilament-200 (NF-200) or glial fibrillary acidic protein (GFAP). The next day, the sections were washed thrice with PBS, and incubated for 1 h with the secondary antibody: goat anti-mouse antibody (DyLight 488 AffiniPure, Earthox Biotechnology, San Francisco, CA, USA). Excess secondary antibody was then rinsed off. After the glass slides were covered with DAPI (4′, 6-diamidino-2-phenylindole), coverslips were placed. After 10 min, the labelled sections were immediately observed with a fluorescence microscope (IX70, Olympus).
Hind-limb motor function evaluation Two researchers who were blind to the group information independently evaluated hind-limb motor function of all the rats using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale [3]. After transplantation, the averaged BBB scores were recorded on days 1, 3, and 7, and then every week up to the fifth week. Tissue sample harvest The rats were deeply anaesthetised with an intraperitoneal injection of pentobarbital sodium (100 mg/kg), then perfused intracardially with 100 mL normal saline (NS) followed by 4 % paraformaldehyde. The spinal cord tissues were dissected, post fixed overnight in 4 % paraformaldehyde, and then successively transferred to 10 and 20 % sucrose solutions overnight. For western blotting, the spinal cords were freshly harvested without perfusion. Each spinal cord was cut into a 10 mm-long block with the centre at the lesion site. The obtained tissues were frozen and cut into 20-μm-thick and 5-μm-thick longitudinal sections using a cryostat (CM3050, Leica, Wetzlar, Germany). Cystic cavity measurement The 5-μm-thick longitudinal sections were fixed in cold acetone for 30 min. The slides were sequentially put into xylene, alcohol, and distilled water. The slides were stained with haematoxylin and rinsed, then put into acid alcohol. After they were counterstained in eosin and rinsed, the slides were taken through distilled water, alcohol, and xylene. The glass slides were air-dried and coverslipped with neutral balsam. Then the area of cystic cavity in the injured spinal cord was measured with a phase-contrast microscope (E100, Nikon, Tokyo, Japan).
Statistical analysis All data are expressed as the mean ± standard deviation (SD). The BBB score data were analysed with repeated measures analysis of variance (ANOVA). For cavity size, the mean protein levels (NT-3, BDNF, VEGF, NF200, and GFAP) were analysed with one-way ANOVA to identify significant differences among the three groups. Statistical significance was inferred when p 95 % of NT-3-DsRed BMSCs and > 99 % of DsRed BMSCs cells exhibited red fluorescence in the cytoplasm (Fig. 1). Because both NT-3 and DsRed genes were transduced into BMSCs and translated from the same mRNA, detection of red fluorescence in NT-3-DsRed BMSCs indicated that these cells expressed NT-3. We subsequently examined NT-3 protein levels by western blot analysis (Fig. 1). A high level of NT-3 was detected in the conditioned medium of NT-3-DsRed BMSCs, whereas no NT-3 was detected in the media of DsRed BMSCs or uninfected BMSCs. Cystic cavity area in rats with induced SCI As the cystic cavity area can vary depending on the section, the ratio of the maximum width of the cavity and the width of the spinal cord on the same dorso-ventral axis was calculated in sagittal cryostat sections stained with haematoxylin–eosin
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Acta Neurochir (2014) 156:1409–1418
Fig. 1 Transfection of NTF3DsRed into BMSCs with lentivirus (a, b) and western blotting detection of NT-3 protein levels (c). a Before transfection. b After transfection. c Lane 1, NTF3-DsRed-BMSC; lane 2, BMSC; lane 3, DsRed-BMSC. Magnification×100 (a, b). Scale bar, 100 μm (a, b)
(HE). The ratio was used to represent the size of the cystic cavity and expressed as a percentage. At 6 weeks after transplantation, the mean values of the ratios in the NT-3-BMSCs, BMSCs, and control groups were 41.29±8.61 %, 58.18± 9.22 %, and 80.84±3.09 % (p
EXPERIMENTAL RESEARCH - BRAIN INJURY
Transplantation of neurotrophin-3-expressing bone mesenchymal stem cells improves recovery in a rat model of spinal cord injury Ling-Jie Wang & Rui-Ping Zhang & Jian-Ding Li
Received: 10 January 2014 / Accepted: 27 March 2014 / Published online: 18 April 2014 # Springer-Verlag Wien 2014
Abstract Background This study aimed to investigate the therapeutic effects of transplanting neutrophin-3 (NT-3)-expressing bone marrow-derived mesenchymal stem cells (BMSCs) in a rat model of spinal cord injury (SCI). Methods Forty-eight adult female Sprague–Dawley rats were randomly assigned to three groups: the control, BMSC, and NT-3-BMSC groups. BMSCs were infected with NT-3DsRed or DsRed lentivirus and injected into the cerebrospinal fluid (CSF) via lumbar puncture (LP) 7 days after SCI in the NT-3-BMSC and BMSC groups, respectively. The hind-limb motor function of all rats was recorded using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale on days 1, 3, 7, 14, 21, 28, and 35 after transplantation. Haematoxylineosin (HE) staining, immunofluorescence labelling, and western blotting were performed at the final time point. Results Expressions of NT-3, brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) proteins increased significantly in the NT-3-BMSC group, and hind-limb locomotor functions improved significantly in the NT-3-BMSC group compared with the other two groups. The cystic cavity area was smallest in the NT-3-BMSC group. In the NT-3-BMSC group, neurofilament 200 (NF200) and glial fibrillary acidic protein (GFAP) expression levels around the lesions were significantly increased and decreased, respectively.
L.DsRed Express2) were constructed and then packaged into lentiviruses with three helper plasmids (pLV/ helper-SL3, pLV/helper-SL4, and pLV/helper-SL5) in 293 T cells by Cyagen Biosciences Inc. (Suzhou, China). The dilution method of plate counting test revealed that the titres of the NTF3-DsRed lentivirus and DsRed lentivirus ranged from 1– 1.8×108 TU/mL and 3.5−3.8×108 TU/mL, respectively. The BMSCs (2×105) in passage 3 were infected with NTF3DsRed lentivirus or DsRed lentivirus following the standard procedures of infection. NTF3-DsRed BMSCs and DsRed BMSCs were harvested, and NTF3-DsRed gene and DsRed gene expressions were detected using a fluorescence microscope (IX71, Olympus, Tokyo, Japan). NTF3 protein was detected by western blotting.
BMSC isolation BMSCs were isolated from the tibias and femurs of 8-weekold female Sprague–Dawley rats (Animal Centre of Chinese People’s Army Military Medical and Scientific Academy, Beijing, China, SCXK2007-004). Donor rats were killed with intraperitoneal injections of an overdose of pentobarbital sodium, and the tibia and femur were dissected. After scraping off all the attached muscles and cutting of both ends of the bones, the marrow was obtained by flushing the bone marrow cavity with 5 mL Dulbecco’s modified essential medium (DMEM; Hyclone, Logan, UT, USA) using a 25-gauge needle. The collected bone marrow was made into a single-cell suspension by triturating with a pipette, and then filtered through a 100-μm mesh nylon cell strainer. After the suspension was centrifuged at 1,500 rpm for 10 min at room temperature, the supernatant was removed. The obtained marrow cells were plated at a cell density of 109/L in 25-cm2 culture flasks in DMEM containing 10 % foetal bovine serum
Preparation of the SCI model Adult female Sprague Dawley rats (8–10 weeks old, weighing 180–240 g) were anaesthetised with an intraperitoneal injection of pentobarbital sodium (50 mg/kg), and laminectomy was performed microscopically on the T7−8 vertebrae. The SCI model was generated using a weight-drop device [6]. A metal rod with a weight of 25 g and diameter of 2 mm was dropped from a height of 3 cm onto the exposed spinal cord to induce a contusion lesion. The twitching hind limbs of the rats completely relaxed, indicating that the SCI model was successful. Then, the muscles and skin were tightly sutured in layers. After surgery, 2×105 U penicillin was given via intramuscular injection every day to prevent infection, and manual emptying of the bladder was performed twice a day until recovery of the micturition reflex. Rats without hind-limb paralysis were excluded.
Acta Neurochir (2014) 156:1409–1418
1411
Group allocation and cell transplantation
Immunofluorescence labelling
A week after contusion, 48 rats were randomly assigned to the following three groups (n=16/group): the control, BMSC, and NT-3-BMSC groups. The rats were re-anaesthetised, and cell transplantation via lumbar puncture (LP) was performed as described previously [1]. In brief, at the L4–L5 intervertebral space, a LP needle (25 gauge) was advanced into the subarachnoid space, and 1×106 cells diluted in 40 μL culture medium were injected into the cerebrospinal fluid (CSF) with a microsyringe. In the control group, the rats received 40 μL culture media without any media or cells via the CSF. For the BMSC and NT-3-BMSC groups, the rats received BMSCs and NT-3-BMSCs, respectively. After cell transplantation, each rat was kept on a 30° slope in the head-down position for 30 min [31].
The 5-μm-thick longitudinal frozen sections were fixed in cold acetone for 30 min. The sections were treated with 0.3 % Triton X-100 in phosphate-buffered saline (PBS) for 30 min at room temperature and immunoblocked with 10 % goat serum in PBS for 1 h at room temperature. Then, they were incubated overnight at 4 °C with the primary antibodies: neurofilament-200 (NF-200) or glial fibrillary acidic protein (GFAP). The next day, the sections were washed thrice with PBS, and incubated for 1 h with the secondary antibody: goat anti-mouse antibody (DyLight 488 AffiniPure, Earthox Biotechnology, San Francisco, CA, USA). Excess secondary antibody was then rinsed off. After the glass slides were covered with DAPI (4′, 6-diamidino-2-phenylindole), coverslips were placed. After 10 min, the labelled sections were immediately observed with a fluorescence microscope (IX70, Olympus).
Hind-limb motor function evaluation Two researchers who were blind to the group information independently evaluated hind-limb motor function of all the rats using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale [3]. After transplantation, the averaged BBB scores were recorded on days 1, 3, and 7, and then every week up to the fifth week. Tissue sample harvest The rats were deeply anaesthetised with an intraperitoneal injection of pentobarbital sodium (100 mg/kg), then perfused intracardially with 100 mL normal saline (NS) followed by 4 % paraformaldehyde. The spinal cord tissues were dissected, post fixed overnight in 4 % paraformaldehyde, and then successively transferred to 10 and 20 % sucrose solutions overnight. For western blotting, the spinal cords were freshly harvested without perfusion. Each spinal cord was cut into a 10 mm-long block with the centre at the lesion site. The obtained tissues were frozen and cut into 20-μm-thick and 5-μm-thick longitudinal sections using a cryostat (CM3050, Leica, Wetzlar, Germany). Cystic cavity measurement The 5-μm-thick longitudinal sections were fixed in cold acetone for 30 min. The slides were sequentially put into xylene, alcohol, and distilled water. The slides were stained with haematoxylin and rinsed, then put into acid alcohol. After they were counterstained in eosin and rinsed, the slides were taken through distilled water, alcohol, and xylene. The glass slides were air-dried and coverslipped with neutral balsam. Then the area of cystic cavity in the injured spinal cord was measured with a phase-contrast microscope (E100, Nikon, Tokyo, Japan).
Statistical analysis All data are expressed as the mean ± standard deviation (SD). The BBB score data were analysed with repeated measures analysis of variance (ANOVA). For cavity size, the mean protein levels (NT-3, BDNF, VEGF, NF200, and GFAP) were analysed with one-way ANOVA to identify significant differences among the three groups. Statistical significance was inferred when p 95 % of NT-3-DsRed BMSCs and > 99 % of DsRed BMSCs cells exhibited red fluorescence in the cytoplasm (Fig. 1). Because both NT-3 and DsRed genes were transduced into BMSCs and translated from the same mRNA, detection of red fluorescence in NT-3-DsRed BMSCs indicated that these cells expressed NT-3. We subsequently examined NT-3 protein levels by western blot analysis (Fig. 1). A high level of NT-3 was detected in the conditioned medium of NT-3-DsRed BMSCs, whereas no NT-3 was detected in the media of DsRed BMSCs or uninfected BMSCs. Cystic cavity area in rats with induced SCI As the cystic cavity area can vary depending on the section, the ratio of the maximum width of the cavity and the width of the spinal cord on the same dorso-ventral axis was calculated in sagittal cryostat sections stained with haematoxylin–eosin
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Acta Neurochir (2014) 156:1409–1418
Fig. 1 Transfection of NTF3DsRed into BMSCs with lentivirus (a, b) and western blotting detection of NT-3 protein levels (c). a Before transfection. b After transfection. c Lane 1, NTF3-DsRed-BMSC; lane 2, BMSC; lane 3, DsRed-BMSC. Magnification×100 (a, b). Scale bar, 100 μm (a, b)
(HE). The ratio was used to represent the size of the cystic cavity and expressed as a percentage. At 6 weeks after transplantation, the mean values of the ratios in the NT-3-BMSCs, BMSCs, and control groups were 41.29±8.61 %, 58.18± 9.22 %, and 80.84±3.09 % (p
