Biotinylated dextran amine tracing of nerve tracts determines regeneration of corticospinal tracts after neural stem cell transplantation＊☆

Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China

lNTRODUCTlON

Nerve tract tracing techniques are an extremely important morphological research method in the field of neural development,function and repair.Horseradish peroxidase(HRP) is a traditional tracer of nerve tracts,and biotinylated dextran amine (BDA) is a new anterograde and retrograde tracer,which visualizes the structures of long conduction tracts and fine terminals[1-2].This technique has been applied in a number of studies[3-4].BDA possesses a stable molecular structure, good water-solubility, no toxicity (no injury to labeled neurons) and relatively static conditions compared with that of HRP[5].BDA is absorbed by neurons and their processes in nerve tissue, then transfers anterogradely or retrogradely to the target area along axons.BDA tracing is visualized by various immunohistochemical techniques for observation under light and electron microscopes[5-6].Our study uses BDA tracing after neural stem cell (NSC)transplantation to provide an advanced,objective and reliable assessment of the morphological changes after NSC transplantation into spinal cord injuries.

RESULTS

Quantitative analysis of experimental animals

Sixteen Sprague-Dawley rats were equally divided into either spinal cord injury or NSC transplantation groups.Both ends of the injured spinal cord were transplanted with autologous NSCs in DMEM/F12 medium immediately after spinal cord transection at the thoracic 10 (T10) segment.One rat in the spinal cord injury group died of a urinary tract infection, and one rat in the NSC transplantation group died of hematuria.The remaining 14 rats were included in the final analysis.

NSC transplantation improves motor function in rats with a T10 spinal cord injury

All rats were healthy prior to experimentation.Bilateral posterior limbs showed complete paralysis after spinal cord transaction and scored 0 points based on the Basso,Beattie and Bresnahan (BBB) locomotor rating scale.BBB scores remained at ≤ 4 points in the two groups with no significant difference after 3 weeks of injury.BBB scores gradually increased at 3 weeks after NSC transplantation and reached ≥ 10 points at 8 weeks, suggesting motor function recovery.The motor function of bilateral posterior limbs in rats in the spinal cord injury group remained unchanged after 12 weeks (Table 1).

BDA anterograde-labeling visualizes NSC transplantation and corticospinal tract recovery in spinal cord injured rats

The corticospinal tract was anterogradely labeled with a cortical five-point pressure injection method and the corticospinal tract was clearly visualized at 2 weeks after labeling.

BDA-labeled cells were located among cortex pyramidal cells in the sensory-motor region.BDA-labeled positive cells were found in pyramidal cells of sensorimotor area cortex, they elicited corticospinal tract traversion in the internal capsule, pontine base and medullary cone, then crossed the posterior funiculus of the spinal cord until the sacral spinal cord (Figure 1).

Table 1 Basso, Beattie and Bresnahan scores of rats after spinal cord injury (score)

Figure 1 Biotinylated dextran amine (BDA) anterogradelabeled corticospinal tract.V: Ventral spinal cord; D: dorsal spinal cord.

In the spinal cord injury group, the corticospinal tract was suspended at the injured segment and distant from the lesions.There were no BDA-labeled corticospinal tract fibers at the transection and distal loci (Figure 2).

Figure 2 Biotinylated dextran amine-labeled corticospinal tract terminates at the injured segment in the spinal cord injury group.

In the NSC transplantation group, the BDA-labeled corticospinal tract crossed through the transection loci and extended toward the distal end in six out of seven rats.Axons regenerated at the cross-sectional level, which showed numerous branches and bead-like nodules that were similar to pre-synaptic vesicles (Figure 3).

Figure 3 A proportion of biotinylated dextranamine-labeled corticospinal tract axons regenerate through the injured segment of the spinal cord in the neural stem cell transplantation group

BDA nerve tract tracing combined with electron microscopy showed that axons swelled, myelin contours were undefined and mitochondrial swelling and crista disappearance was not apparent in the distal end of the spinal cord injury group.BDA-labeled synaptic terminals were not present in all rats.Electron microscopy examination revealed new synaptic connections between regenerated BDA-labeled nerve terminals and host neurons in the NSC transplantation group (Figure 4).

Figure 4 Biotinylated dextran amine (BDA) nerve tract tracing combined with electron microscopy clearlyvisualizes the synaptic terminal of nerve fiber structures(× 10 000).

DlSCUSSlON

Spinal cord repair depends on regeneration of the central nervous system, and an increasing number of studies have shown that cell transplantation into the appropriate microenvironment promotes regeneration[7-11].However,the axonal regeneration mechanism is not yet completely characterized.NSC transplantation contributes toward motor function recovery in animals with spinal cord injury[12-13].At 8 weeks after spinal cord transection, BBB scores of control rats were less than 4 points, while those of NSC transplanted rats were more than 10 points, indicating that NSC transplantation promotes motor function recovery in the posterior limbs of rats.The mechanism underlying NSC transplantation promoting functional recovery after spinal cord injury relies on the transplanted cells themselves and/or neurotrophic factors that stimulate host cell secretion of neurotrophic factors and NSC-derived neurons in the local microenvironment for neural pathway reconstruction and functional recovery[14-16].

Voluntary movement recovery after spinal cord injury largely depends on repair and regeneration of the corticospinal tract.It is unclear whether motor function recovery in rats indicates a proportion of axons regenerated through the transection loci and re-established neural connections after NSC transplantation.Nerve growth and guidance in the corticospinal tract are regulated by cortical motor neurons and depends on the axoplasmic flow that transfers nutrients from the neuron body to maintain nerve metabolism and physiological functions.Therefore, this study used BDA tracer technology after NSC transplantation to investigate the morphology of nerves following spinal cord injury.BDA anterograde-labeled nerve tissues were stained with a free-floating method and were developed with diamino benzidine, which clearly visualizes travelling regions of the corticospinal tract.Neurons and processes were positively stained at the BDA injection site in the cerebral cortex.BDA-labeled corticospinal tract in the brainstem travelled along the ipsilateral injection loci, then after pyramidal decussation, the corticospinal tract travelled in the contralateral spinal cord segments.This morphological assessment is more accurate and reliable compared with that of previous methods that only observe animal behavior to functionally assess and indirectly determine repair of the central nervous system.

This study was able to perform ultrastructural observations due to the highly sensitive characteristics of BDA.Electron microscopy found that a proportion of BDA-labeled axon terminals were visible in the distal end of the injured segment and formed synaptic connections with neuronal processes.This result confirmed that the synaptic-like terminal structure and neurons at the distal end of the injured segment form functional synaptic connections that are related to motor function recovery post-injury.We speculate that in spinal cord injury repair,if BDA-labeled corticospinal tract fibers cross through the transected area, these fibers are the regenerated nerve fibers.BDA nerve tract tracing provides anatomical and morphological evidence for NSC transplantation to treat spinal cord injury for the recovery of hindlimb motor function.

MATERlALS AND METHODS

Design

Randomized controlled animal experiments.

Time and setting

Experiments were conducted from October 2009 to December 2010 at the Institute of Brain and Spinal Cord Injury in Central South University, the Central Laboratory and Neurosurgery Laboratory in Xiangya Hospital, China.

Materials

One healthy Sprague-Dawley rat at gestational day 15,and sixteen adult Sprague-Dawley rats aged 16 months,of either sex and weighing 100–150 g were provided by the Experimental Animal Center of Central South University, China, with license No.SCXK (Hunan)2006-0002.All animal experiments were performed in accordance with theGuidance Suggestions for the Care and Use of Laboratory Animals,issued by the Ministry of Science and Technology of China[17].

Methods

Isolation, identification and collection of NSCs

The rat at gestational day 15 was anesthetized by ether inhalation and fetal mice were collected after laparotomy.Tissues were isolated from the cortex,hippocampus and subependymal zone[18-19], then cut into pieces and placed in a centrifuge tube.The supernatant was removed after centrifugation and the cells were resuspended in DMEM/F12 medium (Gibco,Carlsbad, CA, USA).Cells were centrifuged and the supernatant was removed, DMEM/F12 serum-free complete medium was added, then cells were triturated and filtrated, and the single cell suspension was placed into culture bottles for suspension culture.Half culture medium volumes were exchanged every 2–3 days.Cells were subcultured at 7–9 days and passage 2 cells were cultured for 7–8 days.Cell clones were analyzed for nestin expression by immunofluorescence staining[20-21].Differentiated NSCs were also identified[22-23].Passage 3 NSCs were triturated with a pasteurized pipette to prepare a 2 × 104cells/μL suspension that was stored until use.

Establishment of spinal cord transection injury models

Sixteen Sprague-Dawley rats were intraperitoneally anesthetized with 10% chloral hydrate and fixed in a prone position.The surgical area hair was removed, and the skin was disinfected.Skin and muscle were incised,the vertebral plate was exposed under a microscope(Leica, Heidelberg, Germany), the spinal canal was opened and the spinal cord was exposed and cut with micro-scissors[24-27].Bleeding was stopped with cotton.

NSC transplantation

NSCs were transplanted immediately after the spinal cord was completely transected.The NSC transplantation group was injected with a 2.5 μL NSC suspension into the bilateral ends of the spinal cord, while the spinal cord injury group received 2.5 μL DMEM/F12.After transplantation, the spinal cord surface was dressed with gauze (Surgicel) to stop bleeding, and muscle and skin were closed with a layered suture.Artificial urination was conducted every 8 hours until rats resumed automatic micturition.Rats survived for 3 months post-surgery.

Motor function score

Rats were assessed with the BBB scale every week after spinal cord injury[2,28]to analyze the changes in hindlimb motor function.

BDA nerve tract tracing of the corticospinal tract

Rats were anesthetized at 10 weeks after NSC transplantation and fixed in a stereotaxic apparatus (STOELTING, Wood Dale, IL, USA).The cortical sensorimotor area (0.8 mm posterior to the bregma, 2.2 mm lateral and 1.5 mm deep below the cortex) and adjacent loci (at 2 mm intervals) were injected at five points with a 10%BDA solution (2 μL; Molecular Probes, Carlsbad, CA,USA) using a 5 μL micro-syringe (Shanghai Anting Instrument Factory, China) for 2 weeks antegrade labeling.Then, rats under deep anesthesia were perfused with 4%paraformaldehyde after left ventricular cannulation to the ascending aorta.The brain and spinal cord were immediately removed after perfusion and fixed in 4% paraformaldehyde.Tissues were fixed in a sucrose gradient solution and cut into continuous cross-sectional and sagittal slices with a 30 μm thickness using a freezing microtome (976C type; AO Company, USA).Slices were stained with a free-floating method[29-34]and labeled with an avidin-HRP solution (Molecular Probes) for 2 hours,rinsed, then immersed in a 0.05% DAB solution (Molecular Probes) for 5–10 minutes.Slices were mounted on polylysine coated slides followed by gradient ethanol dehydration and xylene transparency with a water-soluble reagent.

Electron microscopy

Transected spinal cord tissues were fixed in 2.5% paraformaldehyde and glutaraldehyde for 24 hours, then freeze/thawed three times in pentane and liquid nitrogen to increase penetration into the tissue.Specimens were fixed in 1% osmium tetroxide, dehydrated with an acetone gradient and embedded in Epon812 epoxy resin.Slices were trimmed and cut into ultra-thin slices.Cells were reacted with an avidin-HRP solution for 2 hours and colored with a 0.05% DAB solution for 5–10 minutes,then stained with a saturated uranyl acetate and lead nitrate solution for transmission electron microscopy(H-600 type; Hitachi, Tokyo, Japan) observation.

Statistical analysis

Experimental data were analyzed by SPSS 12.0 software (SPSS, Chicago, IL, USA) using pairedt-tests.P＜0.01 was considered statistically significant.

Author contributions:Tao Song and Jun Wu were responsible for data acquisition and integration.Mingyu Zhang proposed and designed this study.Jinfang Liu analyzed experimental data.Jun Wu was responsible for funds and writing the manuscript.Fenghua Chen provided technical and information support.Jiasheng Fang supervised this study.

Conflicts of interest:None declared.

Funding:This study was financially supported by Hunan Province Science and Technology Department Plan, No.2009JT3051.

Ethical approval:The study was approved by the Animal Ethics Committee, Xiangya Hospital, Central South University,China.

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