Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 59, Num. 4, 2011, pp. 579-585

Neurology India, Vol. 59, No. 4, July-August, 2011, pp. 579-585

Topic of the Issue: Original Article

Combination of NEP 1-40 infusion and bone marrow-derived neurospheres transplantation inhibit glial scar formation and promote functional recovery after rat spinal cord injury

Zhou Zhilai¹, Zhang Hui¹, Chen Yinhai², Chen Zhong¹, Min Shaoxiong¹, Yu Bo¹, Jin Anmin¹

¹ Department of Orthopedics, Zhujiang Hospital, Southern Medical University, Guangzhou, China
² Department of Rehabilitation, Zhujiang Hospital, Southern Medical University, Guangzhou, China
Correspondence Address: Jin Anmin, Department of Orthopedics, Zhujiang Hospital, Southern Medical University, 253 Gongye Road, Guangzhou 510282, China, jinanmin2008@yahoo.cn

Date of Submission: 20-May-2011
Date of Decision: 29-Jun-2011
Date of Acceptance: 28-Jul-2011

Code Number: ni11174

PMID: 21891937

DOI: 10.4103/0028-3886.84341

Abstract

Neural stem cell (NSCs) transplantation is another promising strategy for SCI. ^[9] The grafted cells could replace the lost or damaged cells, provide trophic support for neurons, manipulate the environment within the damaged spinal cord to facilitate axon regeneration or promote the plasticity of the injured spinal cord. ^[10],[11] Bone marrow stromal cells (BMSCs) are a heterogeneous population of stem/progenitor cells. ^[12] BMSCs could be induced to NSCs grown as neurospheres under appropriate experimental conditions in vitro. ^[13] In fact, previous experimental studies have shown that neurospheres derived from BMSCs should be an ideal candidate for treatment in SCI as there can be no immunorejection and are easily sourced. ^[14]

In the present study, we combined NEP1-40 with BMSC-NSs transplantation in a SCI rat model. In order to evaluate the possible synergistic effects of NEP1-40 and BMSC-NSs transplantation, we compared combined (BMSC-NSs + NEP1-40) treatment with single-agent treatments (NEP1-40 only, BMSC-NSs only) and with a control group of untreated rats.

Material and Methods

Preparation of bone marrow stromal cells

Isolation of adult rat BMSCs was performed according to a protocol described previously. ^[15] In brief, rats were killed with an overdose of pentobarbital, the marrow was extracted with 10 ml of DMEM/F12 and cultured in a DMEM/F12 basal medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, incubated at 37°, humidity 95% and CO ₂ 5%. After 48 h, the non-adherent cells were removed by changing the medium. BMSCs were subcultured for three to five passages in medium for further analysis. The phenotypic characteristics of BMSCs was detected as follows: cells at passage 4 were trypsinized into single-cell suspensions and incubated with fluorescein (FITC)- or phycoerythrin (PE)-coupled antibodies, including mouse anti-CD45-FITC, CD29-PE, CD34-PE and CD44-PE (ABCAM) at a temperature of 4°C for 30 min. Non-specific anti-rat IgG-FITC and IgG-PE (PHARMINGEN) under the same cultured condition were used as an isotype control, and then flow cytometric analysis was performed.

Neurosphere formation and expansion

For neural induction, BMSCs were dissociated with 0.25% trypsin/0.02% EDTA and plated into a 75 cm ² plastic flask at a concentration of 1×10 ⁵ cells/ml in DMEM/F12 serum-free medium supplemented with 20 ng/ml basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill,NJ,USA), 20 ng/ml epidermal growth factor (EGF; Peprotech) and N2 (1:100; Gibco, BRL, Gaithersburg, MD, USA). The medium and growth factors were added every 2 days. The third passage of neurospheres was used for transplantation. To evaluate the neural differentiation ability of BMSC-derived spheres, BMSC-NSs were plated onto 8-well chamber slides in DMEM/F-12 medium supplemented with 1% N2 supplement, 1% FBS, 5% horse serum (Gibco) and 1 μmol/L retinoic acid (sigma,St. Louis, MO, USA). Cells were differentiated for 6-9 days.

Immunocytochemistry

Immunocytochemistry was performed as described previously. ^[13] The primary antibodies used were rabbit anti-Nestin antibody (1:400; Sigma), mouse anti-CD133 antibody (1:800; Sigma), rabbit anti-MAP2ab antibody (1:800; Sigma) and the secondary antibodies used were FITC-conjugated Goat anti-Rabbit IgG and TRITC-conjugated Goat anti-mouse IgG (1:100; Jackson ImmunoResearch, Baltimore Pike,West Grove,PA,USA). The nuclei were stained using DAPI (Invitrogen, Carlsbad, CA, USA).

Spinal cord surgery

A T10-level contusion SCI model was made according to a protocol described previously. ^[16] Immediately after SCI, a catheter was inserted into the intrathecal space of the spinal cord. The catheter was anchored by suture to the paravertebral muscles and maintained at the lesion site and then was connected to an Alzet osmotic mini pump (model 2004; Alza, Paolo Alto, CA, USA). We adapted the drug concentration and delivery method according to a previous study. ^[8] Intrathecal administration of 500 μM NEP1-40 (Biosynthesis Biotechnology Co. Ltd., Beijing, China) dissolved in 97.5% PBS plus 2.5% DMSO at the rate of 0.25 μl/h was continued for 28 days. The rats were allowed to recover in a warm cage. The antibiotic ampicillin was administered (0.1 g/kg) subcutaneously once a day for 7 days. In addition, manual bladder expression was performed twice daily until spontaneous voiding recovered. Neurological symptoms were assessed with the BBB Locomotor Rating Scale weekly all through the experiment.

Experimental groups

The rats were randomly assigned into four groups. The BMSC-NSs + NEP1-40 group (n=8) received NEP1-40 by an Alzet pump and BMSC-NSs transplantation into the spinal cord; the NEP1-40 only group (n=8) received NEP1-40 by an Alzet pump and PBS injection into the spinal cord; the BMSC-NSs only group (n=8) received PBS by an Alzet pump and BMSC-NSs transplantation into the spinal cord and the control group (n=8) received PBS via an Alzet pump and PBS injection into the spinal cord.

Bone marrow stromal cell-neurospheres transplantation

Before transplantation, bromodeoxyuridine (BrdU, 10 μmol/l; Sigma), a thymidine analog and marker of newly synthesized DNA, was added to the medium for 72 h. Seven days after SCI, the injury site was re-opened, the needle of a 25-μl Hamilton syringe (Hamilton, Reno, NV, USA) was inserted 2 mm into the epicenter of the injury, a total of 10 μl of DMEM/F12 containing 2×10 ⁵ BMSC-NSs was injected into the spinal cord over a period of 5 min and the syringe was left in place for 5 min and then slowly withdrawn to minimize the leakage of cell suspension.

Immunohistochemistry

Rats were sacrificed under deep anesthesia at 10 weeks after SCI and then perfused transcardially. The 10-mm spinal cord segments centered on the compression site were dissected and serial 20-μm longitudinal sections were processed for standard H and E staining and immunostaining. The following antibodies were used as the primary antibody in the immunohistochemical analysis: mouse anti-BrdU (1:200; ABBIOTEC, San Diego, CA, USA), rabbit anti-MAP2ab (1:800; Sigma), rabbit anti-GFAP (1:400; Sigma) and mouse anti-neurofilament (NF, 1:800; Chemicon,Temecula, CA, USA). Samples were examined using a fluorescent microscope (Leica, Germany) and an ImagePro plus 6.0 imaging system. For BrdU ⁺ cell quantification, we used a modified protocol that has been reported to successfully quantitate BrdU labeling. ^[17] Briefly, five slices in every rat and five microscopic visual fields in every slice were selected for analysis. BrdU ⁺ cells were counted at a 400×magnification under the microscope. For double labeling, at least 50 BrdU ⁺ cells per animal were analyzed to confirm the co-localization of the markers for both BrdU and MAP2ab. The intensity of GFAP immunofluorescence was also measured using the ImagePro Plus 6.0 imaging system.

Cavitations analysis

Every eighth section from the spinal cords of rats killed after 10 weeks was processed for H and E staining. The area of cavitations of each section was traced using the Image J software. Any necrotic tissue within the cavities was counted as part of the lesion. The total spinal cord area of the sample was also measured, and included 5 mm rostral and caudal to the injury epicenter for a total of 1 cm length of the spinal cord. The total cavity volume (Vol ^cav ) and total spinal cord volume (Vol ^total ) were calculated using the Cavalieri method. ^[18] The percentage cavitations (%Vol ^cav ) was determined according to the following equation: % Vol ^cav = Vol ^cav /Vol ^total × 100%.

Statistical analysis

All continuous data were expressed as mean ± standard deviation. Differences in all data between groups were evaluated statistically using one-way ANOVA and post hoc Student's t-test. Statistical significance was set at P<0.05.

Results

Characterization of bone marrow stromal cells

The BMSCs harvested from rat bone marrow developed to some elongated cells within primary culture, and there were many small, round cells that were attached to or growing on the BMSCs. These may have been cells from the hematopoietic lineage [Figure - 1]a. The BMSCs became comparatively homogeneous in appearance as the cells passed three to five passages [Figure - 1]b. The FACS analysis demonstrated that the phenotype of the cultured BMSCs was negative for CD34 and CD45 and positive for CD29 and CD44 [Figure - 1]c.

Neurospheres formation

Seven days after induction, large non-adherent NSs were found [Figure - 2]a. Immunocytochemistry revealed that most of the cells within the aggregates were immunopositive for Nestin and CD133, the nuclei were stained by DAPI [Figure - 2]b-e. On further culture, these NSCs were able to differentiate into neuron-like cells [Figure - 2]f.

Combined bone marrow stromal cell-neurospheres transplantation and NEP1-40 infusion reduced the volume of cavity

To elucidate whether the combined therapy would reduce the lesion cavity in injured spinal cord, we measured the area of the cystic cavity with H and E staining 10 weeks after SCI. Quantification analysis showed that there was a significant reduction in the percentages of cavitations in the BMSC-NSs + NEP1-40 group compared with the control group. No significant difference was noted between the other groups [Figure - 3]a.

Combined therapy with NEP1-40 and bone marrow stromal cell-neurospheres transplantation enhanced functional recovery in BBB score after spinal cord injury

All rats became almost completely paraplegic immediately after the injury. Hind limb function had improved significantly in the BMSC-NSs + NEP1-40 group at 7 weeks after injury compared with the control group. The average BBB score in the BMSC-NSs + NEP1-40 group reached 11.0±0.5 at the end of the experiments, which was significantly higher than that in the control group (8.9±0.9) (P<0.05). There was no significant difference between the two single-agent treatment groups and the control group in the BBB score over time [Figure - 3]b.

NEP1-40 enhanced the survival and neural differentiation of bone marrow stromal cell-neurospheres in injured spinal cord

Few BrdU ⁺ grafted cells survived in the injured spinal cord (<2% in the BMSC-NSs + NEP1-40 group and <1% in the BMSC-NSs only group) 9 weeks after transplantation. In the epicenter of the injured spinal cord, some BrdU ⁺ cells could be double-labeled by anti-MAP2ab [Figure - 4]a and b. As shown in [Figure - 4]c, the percentages of BrdU/MAP2ab double-positive cells in the BMSC-NSs + NEP1-40 group reached 0.244±0.015, which was significantly higher than that in the BMSCs-NS only group(0.09±0.028) (P<0.01).

Combined bone marrow stromal cell-neurospheres transplantation and NEP1-40 delivery inhibited the formation of glial scar

Sections double-labeled for GFAP and NF demonstrated that in the BMSC-NSs only group, dense inhibitory GFAP immunoreactivity surrounding the lesion site 10 weeks post-injury. These astrocytes were packed tightly together as a scar barrier and the vast majority of regenerative axons were excluded from the host-lesion interface by this dense scarring [Figure - 5]a-c. However, in the BMSC-NSs + NEP1-40 group, the astrocytes appeared to be permissive and did not form a prominent glial limitans. Axons readily penetrated this barrier and made direct contact with the cystic lesion cavity [Figure - 5]d-f. Fluorescence microscopic analysis showed that the stains of GFAP were significantly lower in the BMSC-NSs + NEP1-40 group than in the BMSC-NSs only group [Figure - 6].

Discussion

The purpose of the study was to determine the efficacy of BMSC-NSs transplantation combined with NEP1-40 on functional recovery after rat SCI. Findings of this study indicated that the combined therapy significantly improved hind limb functional recovery, reduced cystic cavity size and inhibited the formation of glial scars.

The inhospitable microenvironment of the injured spinal cord was unfavorable for the survival and differentiation of NSCs; few NSCs differentiate into neurons after transplantation. ^[19] However, little is known about the exact mechanism of fate decision of NSCs. After the myelin-associated inhibitor Nogo-A was found to be involved in promoting the differentiation of neural progenitors into astroglial lineage cells through the mTORSTAT3 pathway, ^[20] we naturally wondered if blocking the Nogo-A by NEP1-40 would promote the neural differentiation after NSCs transplanted into the injured spinal cord. Our results showed that when BMSC-NSs were transplanted alone, low percentages of cells differentiated into MAP2ab ⁺ neurons. However, when BMSC-NSs transplantation and NEP1-40 were combined, the percentages of cells expressing the neuronal marker MAP2ab significantly improved. Thus, we believe that the combination of NEP1-40 infusion could promote the NSCs differentiated into neurons.

The key strategy of treating SCI by facilitating functional recovery ultimately is to promote axonal regeneration and make the suffering fiber tracts get through the lesion cavity. ^[21] In the present study, the percentages of cavitations was significant smaller in the BMSC-NSs + NEP1-40 group than in the control group; neither NEP1-40 only nor BMSCs-NSs only significantly changed the percentages of cavitations compared with the control group. Immunohistochemistry analysis further demonstrated that in the BMSC-NSs only group, there was a band of glial scar formed by reactive astrocytes at the margin of the injured tissue 10 weeks after SCI, and the axons were completely blocked by the dense barrier, whereas in the BMSC-NSs + NEP1-40 group, the GFAP expression was significantly attenuated and axons readily penetrated this barrier and made direct contact with the cystic lesion cavity. This phenomenon should be ascribed to the biological characteristics of NEP1-40. Because Nogo-A can promote NSCs differentiation into astrocytes, these effects could be blocked by NEP1-40.

The exact mechanisms of functional recovery after cell transplantation are far from clear. Because very few cells survived following grafting, it is unlikely that cellular replacement itself in the injured cord produced improved locomotor recovery. Our result also showed that there was no significant difference in BBB scores between the BMSCs-NSs only group and the control group. Why did the rats in the BMSCs-NSs + NEP1-40 group show a remarkable recovery of locomotive function of hind limbs compared with other groups? The conjectural mechanism may be the synergistic effects of BMSC-NSs and NEP1-40. Because Nogo-A require binding to a complex receptor, including p75 neurotrophin receptor (p75NTR) ^[22] and neurotrophic factors provided by grafted BMSC-NSs are also bind to the p75, ^[23] it suggests that there is a competition for binding to this coreceptor. Thus, blockade of Nogo-A may increase the efficacy of neurotrophins. Several published studies have also reported that local administration of NEP1-40 improved preservation of injured neurons and induced better preservation of neuronal structures following mild cortical injury ^{[4],[8],[24],[25]} and intrathecal administration of NEP1-40 promoted axonal growth and enhanced functional recovery after lateral funiculus injury in the adult rat. ^[8]

In conclusion, combination therapy of BMSC-NSs transplantation and NEP1-40 infusion showed better hind limb functional recovery compared with that seen in the control group,

These effects were not simply the superposition of the two single therapies but may represent a potential new therapy for SCI.

Acknowledgments

This work was supported by a grant from the Natural Science Foundation of Guangdong (8451051501000460).

References

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