Male Sprague-Dawley rats weighing 200-250 g and aged 6-8 weeks were purchased from the Animal Center of the Second Military Medical University. The surgical interventions for animal experiments were approved by by the Ethical Committee of the Shanghai Jiao Tong University School of Medicine, and the animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals after the surgery.
Culture of DRG-NSCs
DRG were dissected from postnatal day 2 rats, mechanically dissociated in Hank’s balanced saline solution, pH 7.4, and seeded in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Invitrogen) supplemented with 2% B27, 10 ng/mL epidermal growth factor (EGF), and 10 ng/mL bFGF. The cells were incubated in a humidified atmosphere with 5% CO2 at 37°C. The medium was changed every 3 days. The dissociated DRG cells formed clusters or neurospheres within 1-2 weeks. Afterwards, the neurospheres were collected, mechanically aspirated, and resuspended in the proliferation culture medium every 4 days to reduce the cell heterogeneity by maintaining small spheres (50-100 cells/sphere). After 3 generations of subcloning, the NSCs derived from the neurospheres were subjected to lentivirus transduction .
Hydrogels were purchased from Beaver Nano-Technologies Co., Ltd, China and were cross-linked with polyethylene glycol diacrylate. Solutions of this hydrogel form a transparent gel when mixed with a cross-linker over a period of 30 minutes. Then, NSCs were mixed with hydrogels to form a stem-cell–hydrogel complex.
Model establishment and NSC transplantation
Forty-eight SD rats were randomly divided into sham, CES model and NSCs transplantation groups. After anesthesia with chloral hydrate, laminectomy was performed at the lumbar 4 and a silicone band (10 mm long, 1 mm wide, and 1 mm thick) was placed under the laminae of the L5-6 vertebra to produce the CES animal model [15,16]. Sixteen rats were subjected to NSC transplantation, and another 16 rats were used as control. A sham operation was performed with a simple laminectomy but without contusion injury (n = 16).
For transplantation of NSCs, we removed the silicone 7 days after the compression injury and then intrathecally injected 12 μL 0.5% hydrogels containing approximately 1000,000 NSCs (transduced with lentivirus) using a micropulled pipette connected to a Hamilton syringe. As control, 12 μL 0.5% hydrogels containing no NSCs was injected into the subarachnoid space.
The cauda equina was extracted, placed in EDTA solution, and heated in an oven for antigen retrieval. Then, 15-μm thick sections of the cauda equina around the lesion site were prepared longitudinally. The tissue sections were permeabilized with 0.2% Triton X-100 and blocked in blocking solution for 1 h at room temperature. After rinsing with PBS, the sections were incubated with primary antibodies against O4, βIII-tubulin, glial fibrillary acidic protein (GFAP) overnight at 4°C followed by incubation with secondary antibodies for 1 h at 37°C. The slices were stained with Hoechst for 10 min and images were photographed by inverted fluorescence microscopy.
For transmission electron microscopic (TEM) studies, the sections were fractured with liquid nitrogen and quenched in hydrogen peroxide solution. After rinsing in PBS, the sections were prepared for ultra-thin sectioning. Tissue sections were fixed in osmium tetroxide, dehydrated in ethanol, and embedded in resin. All samples were observed under transmission electron microscope (TEM).
Luxol fast blue staining
Tissue sections were stained with Luxol fast blue to detect myelin damage as previously described . Briefly, the cauda equina sections were immersed in Luxol fast blue solution in an oven overnight. After rinsing in 95% ethanol and distilled water, the pathological changes of nerve fibers were observed under a light microscope.
TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining
Apoptosis of cauda equina were measured by using a TUNEL detection kit according to the manufacturer’s instructions (Sigma, USA). In brief, paraffin-embedded tissue sections (4-mm-thick) were dewaxed, rehydrated, and incubated with reaction mixture of terminal deoxynucleotidyl transferase for 1 h. After rinsing in PBS, the sections were incubated with biotinylated antibody and ABC complex, and photographed in a light microscope (Zeiss) equipped with a digital camera.
For the tail-flick test, the rats were immobilized 20 min before the test, and the tail was placed over a slit. A beam of light from a projection lamp (voltage of 18.5 V) was focused on the tail skin at the junction between the middle and distal 1/3 of the tail. The latency to respond was recorded with a maximal 15s radiant heat stimulus .
Experimental data are presented as mean ± SD. One-Way ANOVA was used for comparison of different groups. Results were considered statistically significant when the P value was less than 0.05.