- Email: [email protected]
Morphological study of Schwann cells remyelination in contused spinal cord of rats
. 225 .
Chinese Journal of Traumatology 2013;16(4):225-229
Morphological study of Schwann cells remyelination in contused spinal cord of rats LI Yue, ZHANG Lu, ZHANG Jie-yuan, LIU Zheng, DUAN Zhao-xia, LI Bing-cang* 【Abstract】Objective: To study the role and effect of Schwann cells (SCs) remyelination in contused spinal cord. Methods: Green fluorescence protein expressing-SCs were transplanted into the epicenter, rostral and caudal tissues of the injury site at 1 week after the spinal cords were contused. At 6 weeks, the spinal cords were removed for cryosections, semithin sections and ultrathin sections, and then immunocytochemical staining of myelin basic protein (MBP), P0 protein (P0) and S100 protein (S100) was carried out on the cryosections. Qualitative and semiquantitative analyses were performed on the cryosections and semithin sections. Ultrastructure of myelinated fibers was observed on the ultrathin sections under electron microscope. Results: Transplanted SCs and myelinated fibers immunocytochemically labeled by MBP, P0 as well as S100
distributed in whole injured area. The quantity of myelinated fibers labeled by the three myelin proteins showed no statistical difference, however, which was significantly larger than that of controls. On the semithin sections, the experimental group demonstrated more myelinated fibers in the injured area than the controls, but the fibers had smaller diameter and thinner myelin sheath under electron microscope. Conclusion: SCs can promote regeneration of injured nerve fibers and enhance remyelination, which may be histological basis of SCs-mediated functional repair of injured spinal cords. Key words: Spinal cord injury; Schwann cells; Myelin basic protein; Myelin P0 protein; S100 proteins
S
repair experimental spinal cord injury. Compared with olfactory ensheathing cells (OECs) which are often used for repairing different kinds of spinal cord injuries, SCs can not cross glial scar barrier after transplanted into injured spinal cord as well as easily lead to hypertrophy and hyperplasia of astrocyte, and their migration is inhibited when contacting with astrocyte.2 Thus SCs can not migrate and distribute in the spinal cord as widely as OECs can.3,4 Nevertheless, our recent study showed that transplanted SCs like OECs can improve the function of injured spinal cord.5 Hence SCs are still a candidate cell often used as a vector for gene therapy6 and a seed cell for tissue engineering7 in the study of spinal cord injuries. This study aimed at determining effects of transplanted SCs on myelination in injured spinal cords, and providing a histological basis for SCsmediated functional repair of injured spinal cords.
chwann cells (SCs) derived from neural crest, are specific gliocytes in the peripheral nervous system. SCs form myelin sheath of the nerve fibers under normal physiological conditions, while under pathological conditions they induce regeneration of nerve fibers via secreting neurotrophic factors and providing cellular microenvironment for the injured nerve.1 SCs are one of the cells firstly used to
DOI: 10.3760/cma.j.issn.1008-1275.2013.04.008 Neurosurgery Department, 451 Hospital of Chinese People's Liberation Army, Xi’an 710054, China (Li Y) Life School, Southwest University, Chongqing 400715, China (Zhang L) State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing 400042, China (Zhang JY, Duan ZX, Li BC) Ph ysio logical Department, Cho ngqing M edic al University, Chongqing 400042, China (Liu Z) *Corresponding author: Tel: 86-23-68757469, Email: [email protected] This work was supported by grants from the Funds of the State Key Laboratory of Trauma, Burn and Combined Injury (SKLZZ201003) and Special Funds for Major State Basic Research Project, China (2012CB518106).
Chin J Traumatol 2013;16(4):225-229
METHODS Animals Adult Sprague-Dawley rats weighting 200-250 g were housed according to the Third Military Medical University (TMMU, China) guidelines, and surgical procedures
. 226 .
and postoperative care were performed in accordance with protocols approved by TMMU Institutional Animal Care and Use Committee. Water and food were available ad libitum in the cages at all times. Before surgery, rats were sedated with pentobarbital sodium (40 mg/kg, i.p.) and the surgical area was shaved and cleaned with Betadine and 70% alcohol. Rats were kept on a heating pad at 37°C during surgery. Cell culture Simply, sciatic nerves were dissected from threeday-old green fluorescence protein (GFP)-transgenic rats. Tissue was finely minced with scalpel blades on culture dishes and incubated for 30 min in 0.25% trypsin/ 0.02% EDTA digestion solution at 37°C on a rotary shaker in a CO2 incubator. The digestion was stopped by adding DMEM/F12 medium with 20% fetal calf serum, and the pellets were gently triturated with firepolished pasture pipettes. The cell suspension was resuspended and preplated for 1 h in a culture flask at 37°C in a CO2 incubator. Then the cells were centrifuged and resuspended in DMEM/F12. To determine the purity of the SCs, samples were stained for S100 protein (S100), and then covered with coverslip with Citifluor containing Hoechst 33342 to compare the number of S100-positive cells with that of Hoechst-labeled cells. The purity of the SCs was about 95%.8 Spinal cord injury Rats were anesthetized, and a sterile laminectomy was performed at T10 vertebral level without disrupting the dura. The spinal cords were contused using an NYUII impactor with a dropping weight (10.0 g) from 25 mm above the exposed cord. The procedure resulted in hind limb paralysis in all animals. To prevent wound and bladder infections, Bicillin (60 000 U/kg, i.m.) was given daily for a week with bladder compression performed twice daily after contusion. Transplantation of SCs A week after the initial surgery, the rats were reanesthetized and the injured cords were re-exposed. In the experimental group (n=15),1 µl cell suspension (30 000 cells/µl) was injected along the midline of the contused cord at a depth of 0.8 mm of the lesion epicenter and 1 mm rostral and caudal to the epicenter, respectively. A pulled glass micropipette was used in the procedure, which remained in situ for 5 min after each injection. The control group (n=15) were injected
Chinese Journal of Traumatology 2013;16(4):225-229
at identical volumes of DMEM at the same sites. After the injection, the muscle and skin were closed with interrupted sutures. The survival time of all animals was 6 weeks. Histological procedures and semi-quantification At 6 weeks after transplantation, the experimental rats were perfused transcardially with physiological saline, followed by a fixative solution containing 4% paraformaldehyde for cryosections or 2% paraformaldehyde and 2% glutaraldehyde in phosphate buffered saline (PBS) (pH 7.4) for plastic sections. Then, the spinal cords were removed and post-fixed in 4% paraformaldehyde for cryosections or 3% glutaraldehyde for plastic sections. Frozen spinal cords were cut into 15 µm-thickness longitudinal serial sections by Leica cryostat, dried on a heating plate and mounted with Aqua Poly/Mount (Polysciences Inc., USA). The sections were examined using Olympus fluorescence microscope and photographed with DP71 digital camera and software. All lesions were examined in both fluorescein isothiocyanate and rhodamine excitation wavelengths to distinguish between transplanted SCs which fluoresce only in the green wavelength while macrophages autoflouresce in all wavelengths. The digital images were collected at 20× magnification and processed with Adobe Photoshop 10.0. Spot 345 software was used to measure the injuried area and myelinated fibers labeled by myelin basic protein (MBP), P0 protein (P0) and S100 immunoreactivity in the contused cord. Then, percentage of myelin fibers area to injured area was calculated to compare myelination rate between the groups. Plastic semithin sections (1 µm) were collected from osmicated, dehydrated, and embedded tissue blocks. Sections were stained with methylene blue/Azure II and the lesion and location were observed under Olympus BX50 microscope, then photographed at 40× magnification. To count the number of myelinated fibers in the injured area, the sections were taken from every fifth section, with a total of five sections obtained from each animal. Ultrathin sections (0.6 µm) were cut from the plastic-embedded tissue blocks, counterstained with uranyl and lead salts, and examined with a TECNT 10 electron microscope (Netherlands). Immunohistochemistry of MBP, P0 and S100 Longitudinal cryosections were rinsed in PBS for 30 min and then bathed in blocking solution (PBS, 1%
. 227 .
Chinese Journal of Traumatology 2013;16(4):225-229
BSA, 0.3% Triton X-100, 5% normal goat serum) for 1 h at room temperature. Then, the sections were incubated with the primary antibodies: rabbit anti-MBP (1:200, Santa Cruz Biotechnology Inc., USA), rabbit anti-P0 (1:200, Santa Cruz Biotechnology Inc., USA) and mouse anti-S100 (1:200, Chemicon International Inc., USA) monoclonal antibody for 1 h at room temperature and 24 h at 4°C. Then the sections were washed several times with PBS, and incubated with the red fluorescent-conjugated secondary antibody (goat antimouse immunoglobulin G, 1:200, Sigma Chemicals Co., USA) for 2 h at room temperature. Finally, the sections were thoroughly rinsed and embedded in Aqua Poly/Mount (Polysciences Inc., USA). Control sections were systematically included with the test batch where the primary antibody was absent. Statistical analysis Comparison of the myelinated fibers in the injured area was performed using two-tailed Student's t test. Mann-Whitney test was applied to analyze the areas of the f ibers labeled by MBP, P0 and S100 immunoreactivity. P values were rounded to two digits and less than 0.05 was regarded as significant. In order to compare the values between groups, the standard
error was calculated and indicated as ± value in the text.
RESULTS SCs enhancing the expression of three myelin proteins (MBP, P0, S100) and promoting myelination of regenerated nerve fibers In order to study the role and effects of SCs on myelination in contused spinal cords, the expression changes of MBP (Figure 1), P0 (Figure 2) and S100 (Figure 3) were observed by immunocytochemistry. The results showed that three myelin proteins were mainly expressed in healthy tissue of the contused spinal cord with thin cordlike structure crossed together similar with neurofilament-immunoreactive nerve fiber.5 These labeled nerve fibers penetrated into injured area from surrounding healthy tissue with branch-like structures covering the whole injured area. Most of them scattered parallely with SCs and a few overlapped with SCs. The expression of three myelin proteins was found in controls as well, but most only existed along the border between the healthy and injured tissue. In the injured area especially in its center, the labeled nerve fibers were rarely seen (Figure 4).
Figure 1. MBP immunocytochemical reaction in the injured area on cryosections at six weeks after SCs transplantation. A: transplanted GFP-SCs; B: immunolabeled MBP; C: composite image of A and B. The insert in C is the enlargement of “*” labeled region. Figure 2. P0 immunocytochemical reaction in the injured area on cryosections at six weeks after SCs transplantation. A: transplanted GFP-SCs; B: immunolabeled P0; C: composite image of A and B. The insert in C is the enlargement of “*” labeled region. Figure 3. S100 immunocytochemical reaction in the injured area on cryosections at six weeks after SCs transplantation. A: transplanted GFP-SCs; B: immunolabeled S100; C: composite image of A and B. The insert in C is the enlargement of “*” labeled region. Figure 4. MBP (A), P0 (B) and S100 (C) immunocytochemical reactions on cryosections at six weeks after operation in the control group. The labeled nerve fibers can not be observed in the injured area.
The immune reaction products of MBP, P0 and S100 were all red in color. No qualitative difference in number of the nerve fibers labeled by the three myelin proteins
could be recognized among three myelin proteins. No significant difference was observed in the area of the labeled nerve fibers by semi-quantitative analysis (P>0.05)
. 228 .
Chinese Journal of Traumatology 2013;16(4):225-229
with 12.78%, 12.47% and 13.30% of the measured area (0.388 mm2) for MBP-labled, P0-labled and S100-labled fibers, respectively, but the area of labled nerve fibers in the SCs group were significantly greater than that in the control group (2.89% of measured area, P<0.01). SCs increasing myelinated nerve fibers of the injured area and forming myelin sheath Besides cryosections used for studying the labled nerve fibers, plastic sections were also applied to clearly observe role and effects of transplanted SCs on remyelination. Under light microscope, regenerated myelinated fibers could be seen in the area of both the experimental and the control groups on the semithin sections (Figure 5). However, the number of the myelinated fibers in the SCs-receiving animals was more than that in the control group. The results of semiquantitative analysis showed that the number of myelinated nerve fibers was 373.83±63.37 in the transplantation group and only167.33±42.16 in the control group, with significant difference between the two groups (P<0.01). On ultrathin section under electron microscope, it further showed that the number of myelinated nerve fibers in the injured area increased obviously in the transplantation group, and transplanted SCs wrapped the axons to form myelinated fibers, but with smaller diameter and thinner myelin sheath (Figure 6).
Figure 5. Light microscope image of plastic semithin section at six weeks after SCs transplantation. A: the contused spinal cord under low magnification in the control group; B: enlargement of
Figure 6. Electron microscope picture of ultrathin section at six weeks after SCs transplantation. A: myelinated fibers (M) with thick myelin sheath and more nonmyelinated nerve fibers (U) in the control group. B: myelinated fibers (M) with smaller diameter and thinner myelin sheath in the SCs-receiving animals.
DISCUSSION MBP, P0 and S100 are three main structural proteins of nerve myelin sheath. MBP is synthesized by Schwann cells in peripheral nerve system and oligodendroglial cells in central nerve system, occupying about 1/3 of the total myelin protein. This protein can maintain structural and functional stability of myelin sheath, initiate remyelination and promote mitosis of gliocyte. P0 is synthesized only by Schwann cells, hence it is considered as a specific protein of Schwan cells. P0 occupies more than half of the total peripheral nerve myelin protein and plays a key role in maintaining multi-layer structure and compactability of the myelin sheath due to its property of cell adhesion molecules.9 This protein is strongly expressed when remyelination is active, and decreased when the peripheral nerves encountering Wallerian degeneration and demyelination, but increased again when remyelination occurs. S100 is synthesized by oligodendroglial cell and astroglial cell with extensive biological effects such as mediating protein phosphorylation, enzymic activity, Ca2+ balance, cytoskeleton dynamics and inflammatory response. In the nerve system, S100 can promote neurite elongation and enhance activity of developing and injured neuron, so it has a neurotrophic effect.
the frame region in A; C: enlargement of the frame region in B; D: contused spinal cord under low magnification in the transplantation animals; E: enlargement of the frame region in D; F: enlargement of the frame region in E. After transplantation the number of myelinated nerve fibers increased and the tissue was denser and more complete than that of the control group.
Since MBP, P0 and S100 are the main structure proteins of myelin sheath, immuocytochemically labeled structure should be myelin sheath, and the fibers should be myelinated nerve fibers theoretically. Indeed these
. 229 .
Chinese Journal of Traumatology 2013;16(4):225-229
labeled fibers had no difference in morphology with the neurofilament immunoreactive nerve fibers.5 Additionally the electron microscopic observation of this study convinced these fibers to be myelinated nerve fibers. They projected to injured area from healthy tissue, and covered the whole injured area combined with SCs. But in the control group, these myelinated nerve fibers could be seen only along the border between the healthy and injured tissue. It is worthwhile noting that some SCs were parallel with nerve fibers and some overlapped, which indicates that SCs could not only induce nerve fibers to myelin regeneration, but also participate in remyelination themselves. But this study could not demonstrate how many SCs inv olv ing in the remyelination. Besides the transplanted SCs, peripheral SCs were induced by transplanted ones and immigrated centrally, which may also participate in remyelination with oligodendrocytes of spinal cord. Likewise, this study could not determine the proportion of various myelinogenesis cells in remyelination either.
of the injured spinal cord reported by our previous study5 is related with SCs remyelination. In summary, immunocytochemical staining of MBP, P0 and S100 as well as morphological observation indicated that transplanted SCs promoted the regeneration of nerve fibers of contused spinal cords, and enhanced the remyelination by its repairing and inducing other cells. The results may be the histological basis of SCs-mediated functional repair of injured spinal cord.
REFERENCES 1. Plant GW, Currier PF, Cuervo EP, et al. Purified adult ensheathing glia fail to myelinate axons under culture conditions that enable Schwann cells to form myelin. J Neurosci 2002;22 (14):6083-91. 2. Santos-Silva A, Fairless R, Frame MC, et al. FGF/heparin differentially regulates Schwann Cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis. J Neurosci 2007;27(27):7154-67.
Based on the qualitative observation, semiquantitative analysis was carried out in the present study. The results showed no statistical difference in the number of nerve fibers labeled by the three myelin proteins in the experimental group, but the number increased obviously compared with the controls, showing that any of the three proteins can be used for spinal cords injury repair by remyelination of transplanted SCs. Either our semiquantitative data on semithin sections showed that the number of myelinated nerve fibers in the injured area in the experimental group increased significantly compared to the control group, but these fibers had smaller diameter and thinner myelin sheath and basic membrane under electron microscope, indicating that they were new-regenerated nerve fibers.
3. Fairless R, Frame MC, Barnett SC. N-cadherin differentially determines Schwann cell and olfactory ensheathing cell adhesion and migration responses upon contact with astrocytes. Mol Cell Neurosci 2005;28(2):253-63. 4. Andrews MR, Stelzner D. Evaluation of olfactory ensheathing and Schwann cells after implantation into a dorsal injury of adult rat spinal cord. J Neurotrauma 2007;24(11):1773-92. 5. Li BC, Xu C, Zhang JY, et al. Differing Schwann cells and olfactory ensheathing cells behaviors, from interacting with astrocyte, produce similar improvements in contused rat spinal cord's motor function. J Mol Neurosci 2012;48(1):35-44. 6. Blits B, Oudega M, Boer G J, et al. Adeno-associated viral vector–mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function. Neuroscience 2003; 118(1):271-81. 7. Fouad K, Schnell L, Bunge MB, et al. Combining Schwann
After spinal cord injury, it is very difficult for regenerated nerve fibers to enter and pass through the injured area, which is the important reason for incomplete recovery of the function of injured spinal cords. In this study, SCs were transplanted into injured spinal cords. After six weeks all of the nerve fibers labeled by the three myelin proteins entered and covered the injured area concomitant with the transplanted SCs or twisted with each other. Through these observations and the ultrastructure features of myelinated nerve fibers, it is reasonable to judge that improved functional recovery
cell bridges and olfactory-ensheathing glia grafts with chondroitinase promotes locomotor recovery after complete transection of the spinal cord. J Neurosci 2005;25(5):1169-78. 8. You H, Wei L, Liu Y, et al. Olfactory ensheathing cells enhance Schwann cell-mediated anatomical and functional repair after sciatic nerve injury in adult rats. Exp Neurol 2011;229(1): 158-67. (Received January 10, 2013) Edited by DONG Min
Chinese Journal of Traumatology 2013;16(4):225-229
Morphological study of Schwann cells remyelination in contused spinal cord of rats LI Yue, ZHANG Lu, ZHANG Jie-yuan, LIU Zheng, DUAN Zhao-xia, LI Bing-cang* 【Abstract】Objective: To study the role and effect of Schwann cells (SCs) remyelination in contused spinal cord. Methods: Green fluorescence protein expressing-SCs were transplanted into the epicenter, rostral and caudal tissues of the injury site at 1 week after the spinal cords were contused. At 6 weeks, the spinal cords were removed for cryosections, semithin sections and ultrathin sections, and then immunocytochemical staining of myelin basic protein (MBP), P0 protein (P0) and S100 protein (S100) was carried out on the cryosections. Qualitative and semiquantitative analyses were performed on the cryosections and semithin sections. Ultrastructure of myelinated fibers was observed on the ultrathin sections under electron microscope. Results: Transplanted SCs and myelinated fibers immunocytochemically labeled by MBP, P0 as well as S100
distributed in whole injured area. The quantity of myelinated fibers labeled by the three myelin proteins showed no statistical difference, however, which was significantly larger than that of controls. On the semithin sections, the experimental group demonstrated more myelinated fibers in the injured area than the controls, but the fibers had smaller diameter and thinner myelin sheath under electron microscope. Conclusion: SCs can promote regeneration of injured nerve fibers and enhance remyelination, which may be histological basis of SCs-mediated functional repair of injured spinal cords. Key words: Spinal cord injury; Schwann cells; Myelin basic protein; Myelin P0 protein; S100 proteins
S
repair experimental spinal cord injury. Compared with olfactory ensheathing cells (OECs) which are often used for repairing different kinds of spinal cord injuries, SCs can not cross glial scar barrier after transplanted into injured spinal cord as well as easily lead to hypertrophy and hyperplasia of astrocyte, and their migration is inhibited when contacting with astrocyte.2 Thus SCs can not migrate and distribute in the spinal cord as widely as OECs can.3,4 Nevertheless, our recent study showed that transplanted SCs like OECs can improve the function of injured spinal cord.5 Hence SCs are still a candidate cell often used as a vector for gene therapy6 and a seed cell for tissue engineering7 in the study of spinal cord injuries. This study aimed at determining effects of transplanted SCs on myelination in injured spinal cords, and providing a histological basis for SCsmediated functional repair of injured spinal cords.
chwann cells (SCs) derived from neural crest, are specific gliocytes in the peripheral nervous system. SCs form myelin sheath of the nerve fibers under normal physiological conditions, while under pathological conditions they induce regeneration of nerve fibers via secreting neurotrophic factors and providing cellular microenvironment for the injured nerve.1 SCs are one of the cells firstly used to
DOI: 10.3760/cma.j.issn.1008-1275.2013.04.008 Neurosurgery Department, 451 Hospital of Chinese People's Liberation Army, Xi’an 710054, China (Li Y) Life School, Southwest University, Chongqing 400715, China (Zhang L) State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing 400042, China (Zhang JY, Duan ZX, Li BC) Ph ysio logical Department, Cho ngqing M edic al University, Chongqing 400042, China (Liu Z) *Corresponding author: Tel: 86-23-68757469, Email: [email protected] This work was supported by grants from the Funds of the State Key Laboratory of Trauma, Burn and Combined Injury (SKLZZ201003) and Special Funds for Major State Basic Research Project, China (2012CB518106).
Chin J Traumatol 2013;16(4):225-229
METHODS Animals Adult Sprague-Dawley rats weighting 200-250 g were housed according to the Third Military Medical University (TMMU, China) guidelines, and surgical procedures
. 226 .
and postoperative care were performed in accordance with protocols approved by TMMU Institutional Animal Care and Use Committee. Water and food were available ad libitum in the cages at all times. Before surgery, rats were sedated with pentobarbital sodium (40 mg/kg, i.p.) and the surgical area was shaved and cleaned with Betadine and 70% alcohol. Rats were kept on a heating pad at 37°C during surgery. Cell culture Simply, sciatic nerves were dissected from threeday-old green fluorescence protein (GFP)-transgenic rats. Tissue was finely minced with scalpel blades on culture dishes and incubated for 30 min in 0.25% trypsin/ 0.02% EDTA digestion solution at 37°C on a rotary shaker in a CO2 incubator. The digestion was stopped by adding DMEM/F12 medium with 20% fetal calf serum, and the pellets were gently triturated with firepolished pasture pipettes. The cell suspension was resuspended and preplated for 1 h in a culture flask at 37°C in a CO2 incubator. Then the cells were centrifuged and resuspended in DMEM/F12. To determine the purity of the SCs, samples were stained for S100 protein (S100), and then covered with coverslip with Citifluor containing Hoechst 33342 to compare the number of S100-positive cells with that of Hoechst-labeled cells. The purity of the SCs was about 95%.8 Spinal cord injury Rats were anesthetized, and a sterile laminectomy was performed at T10 vertebral level without disrupting the dura. The spinal cords were contused using an NYUII impactor with a dropping weight (10.0 g) from 25 mm above the exposed cord. The procedure resulted in hind limb paralysis in all animals. To prevent wound and bladder infections, Bicillin (60 000 U/kg, i.m.) was given daily for a week with bladder compression performed twice daily after contusion. Transplantation of SCs A week after the initial surgery, the rats were reanesthetized and the injured cords were re-exposed. In the experimental group (n=15),1 µl cell suspension (30 000 cells/µl) was injected along the midline of the contused cord at a depth of 0.8 mm of the lesion epicenter and 1 mm rostral and caudal to the epicenter, respectively. A pulled glass micropipette was used in the procedure, which remained in situ for 5 min after each injection. The control group (n=15) were injected
Chinese Journal of Traumatology 2013;16(4):225-229
at identical volumes of DMEM at the same sites. After the injection, the muscle and skin were closed with interrupted sutures. The survival time of all animals was 6 weeks. Histological procedures and semi-quantification At 6 weeks after transplantation, the experimental rats were perfused transcardially with physiological saline, followed by a fixative solution containing 4% paraformaldehyde for cryosections or 2% paraformaldehyde and 2% glutaraldehyde in phosphate buffered saline (PBS) (pH 7.4) for plastic sections. Then, the spinal cords were removed and post-fixed in 4% paraformaldehyde for cryosections or 3% glutaraldehyde for plastic sections. Frozen spinal cords were cut into 15 µm-thickness longitudinal serial sections by Leica cryostat, dried on a heating plate and mounted with Aqua Poly/Mount (Polysciences Inc., USA). The sections were examined using Olympus fluorescence microscope and photographed with DP71 digital camera and software. All lesions were examined in both fluorescein isothiocyanate and rhodamine excitation wavelengths to distinguish between transplanted SCs which fluoresce only in the green wavelength while macrophages autoflouresce in all wavelengths. The digital images were collected at 20× magnification and processed with Adobe Photoshop 10.0. Spot 345 software was used to measure the injuried area and myelinated fibers labeled by myelin basic protein (MBP), P0 protein (P0) and S100 immunoreactivity in the contused cord. Then, percentage of myelin fibers area to injured area was calculated to compare myelination rate between the groups. Plastic semithin sections (1 µm) were collected from osmicated, dehydrated, and embedded tissue blocks. Sections were stained with methylene blue/Azure II and the lesion and location were observed under Olympus BX50 microscope, then photographed at 40× magnification. To count the number of myelinated fibers in the injured area, the sections were taken from every fifth section, with a total of five sections obtained from each animal. Ultrathin sections (0.6 µm) were cut from the plastic-embedded tissue blocks, counterstained with uranyl and lead salts, and examined with a TECNT 10 electron microscope (Netherlands). Immunohistochemistry of MBP, P0 and S100 Longitudinal cryosections were rinsed in PBS for 30 min and then bathed in blocking solution (PBS, 1%
. 227 .
Chinese Journal of Traumatology 2013;16(4):225-229
BSA, 0.3% Triton X-100, 5% normal goat serum) for 1 h at room temperature. Then, the sections were incubated with the primary antibodies: rabbit anti-MBP (1:200, Santa Cruz Biotechnology Inc., USA), rabbit anti-P0 (1:200, Santa Cruz Biotechnology Inc., USA) and mouse anti-S100 (1:200, Chemicon International Inc., USA) monoclonal antibody for 1 h at room temperature and 24 h at 4°C. Then the sections were washed several times with PBS, and incubated with the red fluorescent-conjugated secondary antibody (goat antimouse immunoglobulin G, 1:200, Sigma Chemicals Co., USA) for 2 h at room temperature. Finally, the sections were thoroughly rinsed and embedded in Aqua Poly/Mount (Polysciences Inc., USA). Control sections were systematically included with the test batch where the primary antibody was absent. Statistical analysis Comparison of the myelinated fibers in the injured area was performed using two-tailed Student's t test. Mann-Whitney test was applied to analyze the areas of the f ibers labeled by MBP, P0 and S100 immunoreactivity. P values were rounded to two digits and less than 0.05 was regarded as significant. In order to compare the values between groups, the standard
error was calculated and indicated as ± value in the text.
RESULTS SCs enhancing the expression of three myelin proteins (MBP, P0, S100) and promoting myelination of regenerated nerve fibers In order to study the role and effects of SCs on myelination in contused spinal cords, the expression changes of MBP (Figure 1), P0 (Figure 2) and S100 (Figure 3) were observed by immunocytochemistry. The results showed that three myelin proteins were mainly expressed in healthy tissue of the contused spinal cord with thin cordlike structure crossed together similar with neurofilament-immunoreactive nerve fiber.5 These labeled nerve fibers penetrated into injured area from surrounding healthy tissue with branch-like structures covering the whole injured area. Most of them scattered parallely with SCs and a few overlapped with SCs. The expression of three myelin proteins was found in controls as well, but most only existed along the border between the healthy and injured tissue. In the injured area especially in its center, the labeled nerve fibers were rarely seen (Figure 4).
Figure 1. MBP immunocytochemical reaction in the injured area on cryosections at six weeks after SCs transplantation. A: transplanted GFP-SCs; B: immunolabeled MBP; C: composite image of A and B. The insert in C is the enlargement of “*” labeled region. Figure 2. P0 immunocytochemical reaction in the injured area on cryosections at six weeks after SCs transplantation. A: transplanted GFP-SCs; B: immunolabeled P0; C: composite image of A and B. The insert in C is the enlargement of “*” labeled region. Figure 3. S100 immunocytochemical reaction in the injured area on cryosections at six weeks after SCs transplantation. A: transplanted GFP-SCs; B: immunolabeled S100; C: composite image of A and B. The insert in C is the enlargement of “*” labeled region. Figure 4. MBP (A), P0 (B) and S100 (C) immunocytochemical reactions on cryosections at six weeks after operation in the control group. The labeled nerve fibers can not be observed in the injured area.
The immune reaction products of MBP, P0 and S100 were all red in color. No qualitative difference in number of the nerve fibers labeled by the three myelin proteins
could be recognized among three myelin proteins. No significant difference was observed in the area of the labeled nerve fibers by semi-quantitative analysis (P>0.05)
. 228 .
Chinese Journal of Traumatology 2013;16(4):225-229
with 12.78%, 12.47% and 13.30% of the measured area (0.388 mm2) for MBP-labled, P0-labled and S100-labled fibers, respectively, but the area of labled nerve fibers in the SCs group were significantly greater than that in the control group (2.89% of measured area, P<0.01). SCs increasing myelinated nerve fibers of the injured area and forming myelin sheath Besides cryosections used for studying the labled nerve fibers, plastic sections were also applied to clearly observe role and effects of transplanted SCs on remyelination. Under light microscope, regenerated myelinated fibers could be seen in the area of both the experimental and the control groups on the semithin sections (Figure 5). However, the number of the myelinated fibers in the SCs-receiving animals was more than that in the control group. The results of semiquantitative analysis showed that the number of myelinated nerve fibers was 373.83±63.37 in the transplantation group and only167.33±42.16 in the control group, with significant difference between the two groups (P<0.01). On ultrathin section under electron microscope, it further showed that the number of myelinated nerve fibers in the injured area increased obviously in the transplantation group, and transplanted SCs wrapped the axons to form myelinated fibers, but with smaller diameter and thinner myelin sheath (Figure 6).
Figure 5. Light microscope image of plastic semithin section at six weeks after SCs transplantation. A: the contused spinal cord under low magnification in the control group; B: enlargement of
Figure 6. Electron microscope picture of ultrathin section at six weeks after SCs transplantation. A: myelinated fibers (M) with thick myelin sheath and more nonmyelinated nerve fibers (U) in the control group. B: myelinated fibers (M) with smaller diameter and thinner myelin sheath in the SCs-receiving animals.
DISCUSSION MBP, P0 and S100 are three main structural proteins of nerve myelin sheath. MBP is synthesized by Schwann cells in peripheral nerve system and oligodendroglial cells in central nerve system, occupying about 1/3 of the total myelin protein. This protein can maintain structural and functional stability of myelin sheath, initiate remyelination and promote mitosis of gliocyte. P0 is synthesized only by Schwann cells, hence it is considered as a specific protein of Schwan cells. P0 occupies more than half of the total peripheral nerve myelin protein and plays a key role in maintaining multi-layer structure and compactability of the myelin sheath due to its property of cell adhesion molecules.9 This protein is strongly expressed when remyelination is active, and decreased when the peripheral nerves encountering Wallerian degeneration and demyelination, but increased again when remyelination occurs. S100 is synthesized by oligodendroglial cell and astroglial cell with extensive biological effects such as mediating protein phosphorylation, enzymic activity, Ca2+ balance, cytoskeleton dynamics and inflammatory response. In the nerve system, S100 can promote neurite elongation and enhance activity of developing and injured neuron, so it has a neurotrophic effect.
the frame region in A; C: enlargement of the frame region in B; D: contused spinal cord under low magnification in the transplantation animals; E: enlargement of the frame region in D; F: enlargement of the frame region in E. After transplantation the number of myelinated nerve fibers increased and the tissue was denser and more complete than that of the control group.
Since MBP, P0 and S100 are the main structure proteins of myelin sheath, immuocytochemically labeled structure should be myelin sheath, and the fibers should be myelinated nerve fibers theoretically. Indeed these
. 229 .
Chinese Journal of Traumatology 2013;16(4):225-229
labeled fibers had no difference in morphology with the neurofilament immunoreactive nerve fibers.5 Additionally the electron microscopic observation of this study convinced these fibers to be myelinated nerve fibers. They projected to injured area from healthy tissue, and covered the whole injured area combined with SCs. But in the control group, these myelinated nerve fibers could be seen only along the border between the healthy and injured tissue. It is worthwhile noting that some SCs were parallel with nerve fibers and some overlapped, which indicates that SCs could not only induce nerve fibers to myelin regeneration, but also participate in remyelination themselves. But this study could not demonstrate how many SCs inv olv ing in the remyelination. Besides the transplanted SCs, peripheral SCs were induced by transplanted ones and immigrated centrally, which may also participate in remyelination with oligodendrocytes of spinal cord. Likewise, this study could not determine the proportion of various myelinogenesis cells in remyelination either.
of the injured spinal cord reported by our previous study5 is related with SCs remyelination. In summary, immunocytochemical staining of MBP, P0 and S100 as well as morphological observation indicated that transplanted SCs promoted the regeneration of nerve fibers of contused spinal cords, and enhanced the remyelination by its repairing and inducing other cells. The results may be the histological basis of SCs-mediated functional repair of injured spinal cord.
REFERENCES 1. Plant GW, Currier PF, Cuervo EP, et al. Purified adult ensheathing glia fail to myelinate axons under culture conditions that enable Schwann cells to form myelin. J Neurosci 2002;22 (14):6083-91. 2. Santos-Silva A, Fairless R, Frame MC, et al. FGF/heparin differentially regulates Schwann Cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis. J Neurosci 2007;27(27):7154-67.
Based on the qualitative observation, semiquantitative analysis was carried out in the present study. The results showed no statistical difference in the number of nerve fibers labeled by the three myelin proteins in the experimental group, but the number increased obviously compared with the controls, showing that any of the three proteins can be used for spinal cords injury repair by remyelination of transplanted SCs. Either our semiquantitative data on semithin sections showed that the number of myelinated nerve fibers in the injured area in the experimental group increased significantly compared to the control group, but these fibers had smaller diameter and thinner myelin sheath and basic membrane under electron microscope, indicating that they were new-regenerated nerve fibers.
3. Fairless R, Frame MC, Barnett SC. N-cadherin differentially determines Schwann cell and olfactory ensheathing cell adhesion and migration responses upon contact with astrocytes. Mol Cell Neurosci 2005;28(2):253-63. 4. Andrews MR, Stelzner D. Evaluation of olfactory ensheathing and Schwann cells after implantation into a dorsal injury of adult rat spinal cord. J Neurotrauma 2007;24(11):1773-92. 5. Li BC, Xu C, Zhang JY, et al. Differing Schwann cells and olfactory ensheathing cells behaviors, from interacting with astrocyte, produce similar improvements in contused rat spinal cord's motor function. J Mol Neurosci 2012;48(1):35-44. 6. Blits B, Oudega M, Boer G J, et al. Adeno-associated viral vector–mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function. Neuroscience 2003; 118(1):271-81. 7. Fouad K, Schnell L, Bunge MB, et al. Combining Schwann
After spinal cord injury, it is very difficult for regenerated nerve fibers to enter and pass through the injured area, which is the important reason for incomplete recovery of the function of injured spinal cords. In this study, SCs were transplanted into injured spinal cords. After six weeks all of the nerve fibers labeled by the three myelin proteins entered and covered the injured area concomitant with the transplanted SCs or twisted with each other. Through these observations and the ultrastructure features of myelinated nerve fibers, it is reasonable to judge that improved functional recovery
cell bridges and olfactory-ensheathing glia grafts with chondroitinase promotes locomotor recovery after complete transection of the spinal cord. J Neurosci 2005;25(5):1169-78. 8. You H, Wei L, Liu Y, et al. Olfactory ensheathing cells enhance Schwann cell-mediated anatomical and functional repair after sciatic nerve injury in adult rats. Exp Neurol 2011;229(1): 158-67. (Received January 10, 2013) Edited by DONG Min