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 basic fibroblast growth factor (bFGF). NSCs were cultured in a 6-well culture plate at a density of 50-100 cells/μL with 5% CO2 at 37°C. The medium was changed 2-3 times a week. The dissociated DRG cells formed clusters or neurospheres within 72 hours. During the passage of neurospheres, the contaminated neurosphere should be removed from each culture hole. The neurospheres and culture medium in the whole culture plate should be transferred to a 15mL centrifuge tube, centrifuged for 200g 5 minutes, and the supernatant should be discarded. 2mL trypsin was added to the cells precipitation. Use Pasteur pipette to gently blow the neurospheres, place it at 37°C for 20 minutes, centrifugation, and discard the supernatant. The precipitation was suspended in a small amount of medium, gently beaten and mixed, counted, and then laid in a 6-well plate, each hole about 200-300 neurospheres .
After 3 generations of subcloning, the NSCs derived from the neurospheres. Some of them were subjected to lentivirus transfection for carrying green fluorescent protein (GFP) .
Hydrogels were purchased from Beaver Nano-Technologies Co., Ltd, China. Hydrogel products are made from polypeptide nanomaterials. After entering the body, the gelation process can be initiated quickly in the damaged part, and then half liquid or solid hydrogel can be formed in about 30 seconds.
Hydrogel pretreatment: The original solution of the hydrogel was treated with ultrasonic water bath for 30 minutes at room temperature to reduce the viscosity of the original solution.
Preparation of cells working solution: the NSCs suspension was centrifuged at low speed to remove the supernatant. 5mL of 10% sterile sucrose solution was added to the collected cells to resuspend the cells. The cells suspension was centrifuged again, and the supernatant was discarded to remove the remaining ionic components in the protocell precipitation. Then, the cells were resuspended with 50μL 10% sterile sucrose solution to prepare salt ion free isotonic cells suspension.
Preparation of hydrogel cells mixture: The 50μL pretreatment hydrogel solution was mixed lightly with 50μL NSCs suspension. Slightly 100μL Phosphate buffer saline was added to the upper layer of the above mixture. Then, the PBS layer and the hydrogel layer were mixed evenly with the pipette, and finally 200μL hydrogel cell mixture was obtained. The final concentration of the hydrogel is 0.25%.
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 12μL 0.25% hydrogels containing approximately 1000,000 NSCs (transfected with lentivirus vectors carrying GFP) using a micropulled pipette connected to a Hamilton syringe. As control, 12μL 0.25% hydrogels containing no NSCs was injected into the subarachnoid space.
The rats were perfused with 4% paraformaldehyde as the fixative. The cauda equina was then 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 hour at room temperature. After rinsing with PBS, the sections were incubated with primary antibodies against O4, βIII-tubulin, glial fibrillary acidic protein (GFAP)，S100 overnight at 4°C followed by incubation with secondary antibodies for 1 hour at 37°C. The slices were stained with Hoechst for 10 minutes and images were photographed by inverted fluorescence microscopy. The staining of NSCs in vitro was the same as the above methods.
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 TEM.
Luxol fast blue staining
Tissue sections were stained with Luxol fast blue (LFB) 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.