brain research 1561 (2014) 1–10

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Research Report

Transplantation of mesenchymal stem cells exerts anti-apoptotic effects in adult rats after spinal cord ischemia-reperfusion injury Fei Yina,1, Li Guob,n, Chun-yang Menga, Ya-juan Liuc, Ri-feng Lub, Peng Lib, Yu-bo Zhoub a

Department of Spine Surgery, First Hospital of Jilin University, Changchun 130021, Jilin, PR China Department of Toxicology, School of Public Health, Jilin University, Changchun 130021, Jilin, PR China c Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Changchun 130021, PR China b

art i cle i nfo

ab st rac t

Article history:

It is unknown whether transplantation of bone marrow mesenchymal stem cells (BM-MSCs)

Accepted 27 February 2014

can repair spinal cord ischemia-reperfusion injury (SCII) in a rat model through an anti-

Available online 5 March 2014

apoptotic effect. Adult rats were divided into untreated or sham-operated controls, untreated models of SCII (uSCII) and BM-MSC-transplanted models of SCII (tSCII; labeled with CM-Dill

Keywords:

transplanted at 1 h and 24 h after reperfusion). According to evaluation of hind-limb motor

Mesenchymal stem cells Spinal cord ischemia-reperfusion injury

function, the motor functions of tSCII rats were significantly better than those of uSCII rats by the seventh day. H&E and TUNEL staining showed that the spinal cords of uSCII rats contained damaged neural cells with nuclear pyknosis and congestion of blood vessels, with a high

Transplantation

percentage of apoptotic neural cells, while the spinal cords of tSCII rats were nearly normal with

Anti-apoptosis

significantly fewer apoptotic neural cells. Immunohistochemistry and double immunofluorescence staining revealed that in tSCII rats CASP3 and neurofilament-H (NF-H) levels were 14.57% and 174% those of uSCII rats, respectively, and in tSCII rats the ratio of BAX to BCL2 was reduced by nearly 50%. The differentiation of transplanted CM-Dil-labeled BM-MSCs into neurons and astrocytes was observed in the spinal cords of the tSCII rats under laser scanning confocal microscopy. These results showed that transplantation of BM-MSCs improved functional recovery after SCII via anti-apoptosis. & 2014 Elsevier B.V. All rights reserved.

Abbreviations:

(BM-MSCs),

bone

marrow

mesenchymal

stem

cells;

(SCII),

(tSCII), transplantation model of spinal cord ischemia-reperfusion injury; (uSCII), n Corresponding author. E-mail addresses: [email protected] (F. Yin), [email protected] (L. Guo). 1 Fax: 86 431 85619418. http://dx.doi.org/10.1016/j.brainres.2014.02.047 0006-8993 & 2014 Elsevier B.V. All rights reserved.

spinal

cord

ischemia-reperfusion

untreated SCII model

injury;

2

1.

brain research 1561 (2014) 1–10

Introduction

Spinal cord ischemia-reperfusion injury (SCII) is a devastating problem that leads to severe complications, including paralysis (Guerit and Dion, 2002). Many researchers have focused on the prevention and treatment of SCII with strategies such as induced hypothermia during surgical procedures (Cakir et al., 2003), ischemic preconditioning (Zvara et al., 1999), cerebrospinal fluid drainage (Cina et al., 2004) and pharmacological interventions with dapsone (Diaz-Ruiz et al., 2011), puerarin (Tian et al., 2011), or mesna (2-mercaptoethane sulfonate sodium) (Dolgun et al., 2010). Nevertheless, definitive prevention and therapy for SCII has yet to be determined. Apoptosis includes both a receptor-dependent extrinsic pathway (Sakurai et al., 1998) and a mitochondria-associated intrinsic pathway (Springer et al., 1999), which are activated in spinal cord injuries. Mackey et al. (1997) and Hayashi et al. (1998) have shown that neuronal apoptosis occurs in a rabbit model of SCII. It has been shown that CASP3 and CASP9, effectors of apoptosis, are involved in mitochondriaassociated apoptotic pathways (Li et al., 1997; Springer et al., 1999). Many other genes are involved in apoptosis, such as p53 (Kotipatruni et al., 2011), the anti-apoptosis gene Bcl2 (Kroemer, 1997; Woo et al., 2005) and apoptosis-inducer Bax (Kotipatruni et al., 2011). It is known that the Bcl2 gene family includes genes for encoding the pro-apoptosis proteins BAX and BAK, and the genes for anti-apoptosis proteins BCL2 and BCLX (Kotipatruni et al., 2011; Kroemer, 1997; Sentman et al., 1991; Woo et al., 2005; Youle and Strasser, 2008). BCL2 is a 26-kDa protein located in mitochondrial membranes; overexpression of BCL2 inhibits apoptosis of neurons (Allsopp et al., 1993; Fan et al., 2010; Kane et al., 1993; Liu et al., 2011; Reed, 1997). Wang et al. (2011) demonstrated that overexpression of BCL2 inhibited neuronal apoptosis after spinal cord injury and improved recovery of neurological function in Bcl2-overexpressed transgenic mice. In contrast, BAX, a cytosolic protein in normal living cells, can induce apoptosis and quickly translocates to mitochondria at an early stage of the apoptotic process (Gross et al., 1998; Hsu and Youle, 1997). The anti-apoptotic effect of BCL2 hinders the activity of the BAX pro-apoptotic protein. The ratio of BAX and BCL2 in the outer membrane of the mitochondrion is a determining factor for the release of cytochrome C (Adams and Cory, 1998), and its binding to cytosolic Apaf-1 (apoptotic protease activating factor 1) and pro-CASP 9 to form complexes activates CASP9, which then activates CASP3 and ultimately initiates apoptosis (Li et al., 1997). Kotipatruni et al. (2011) showed upregulation of phosphop53 and BAX, downregulation of BCL2, and the release of cytochrome C in rat spinal cords after spinal cord injury. These results point to a mitochondrial-mediated apoptotic pathway after spinal cord injury. Further studies have confirmed that the expressions of CASP9 and CASP3 observed after spinal cord injury are intrinsic key mediators of apoptosis (Barut et al., 2005; Kakinohana et al., 2011; Kanellopoulos et al., 1997; Kotipatruni et al., 2011; Yakovlev et al., 1997). Raisova et al. (2001) demonstrated the importance of the ratio of BAX to BCL2 in apoptosis. The ratios in 11 human

melanoma cells were evaluated by Western blot and the results revealed that susceptibility to CD95/Fas-mediated apoptosis in melanoma cells was directly associated with the BAX/BCL2 ratio. Moreover, the results showed that cells resistant to apoptosis had a characteristically low BAX/BCL2 ratio while cells sensitive to apoptosis had a high ratio. A previous study showed the anti-apoptotic effects of transplantation of bone marrow mesenchymal stem cells (BM-MSCs) in acutely-induced Alzheimer's disease mice brains (Lee et al., 2010). Moreover, BM-MSCs can migrate and integrate into damaged organs or tissues (Jin et al., 2013; Sato et al., 2011; Soler et al., 2012; Stamm et al., 2003) where they differentiate into cell lineages such as myocardiocytes (Guan et al., 2011) and neurons (Wei et al., 2012). It remains unclear whether transplantation of BM-MSCs has an anti-apoptotic effect in the injured spinal cord of an animal model of spinal cord ischemia. Therefore, in the present study, we investigated the effects of BM-MSCs on apoptosis of nerve cells after transplantation of BM-MSCs into adult rat with SCII.

2.

Results

2.1.

Establishment of rat BM-MSCs culture

Cells attached to flasks 3 h after seeding. The number of attached cells increased after change of the culture medium on day 3. The majority of cells appeared spindle-shaped, only a few cells were triangular. After two passages, morphologically homogenous populations of fibroblast-like cells were observed. Few of the cultured cells expressed CD45 antigen (1.01%). However, these cells were strongly positive for CD44 (99.35%) and CD90 antigen (99.16%). Results showing BMSCs positive for CD44 and CD90 but negative for CD45 indicated that they were not hematopoietic (Fig. 1).

2.2.

Efficacy of CM-Dil labeled BM-MSCs

Red fluorescence was observed in the cytoplasm of BM-MSCs after the cells were labeled, the percentage of the BM-MSCs labeled by CM-Dil was 90.4% assessed by flow cytometry assay.

2.3. Transplantation of BM-MSCs significantly improved neurological deficit in the animals with SCII To examine effects of BM-MSCs on functional recovery, we used the BBB locomotor rating scale to compare hind-limb motor functions among groups during 7 days immediately after ischemia-reperfusion (Fig. 2). We observed that the hind-limb motor functions were normal in the controls (non-operated and uSCII model; the normal score was 21) and sham-operated groups. During the first 3 days, the animals in both the uSCII model and MSC-transplanted (tSCII) groups scored o14.5 on the BBB scale, indicating that the animals in both these two groups had developed unambiguous neurological deficit. However, on the fourth day, the BBB scores of the animals in the uSCII model group were

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Fig. 1 – Characterization of rat BM-MSCs. The results of cell surface antigen detection showed that BM-MSCs were positive for CD44 (99.35%) and CD90 (99.16%), and negative for CD45 (1.01%).

Fig. 2 – Evaluation of hindlimb motor function. The animals of the uSCII model and cell transplantation (tSCII) groups developed obvious neurological deficit in the first 3 days, however, from the fourth day, the animals in the transplantation group showed a significant improvement in neurologic deficit compared with that in the uSCII model (Po0.05). nPo0.05 compared with the control group; #Po0.05 compared with the sham-operated group; △Po0.05 compared with the uSCII model group.

significantly lower than those of the animals in the tSCII group (Po0.05). From the fourth to the seventh day, the BBB score of the animals in the tSCII group was nearly normal and on the seventh day reached 20.63.

2.4. BM-MSC transplantation reduced morphological damage of nerve cells in the spinal cords of rats with SCII Pathohistological analysis using H&E staining of the spinal cords in the control and sham-operated groups did not reveal any structural or neuronal damage (Fig. 3A). The animals in the uSCII model group showed some injured motor neurons with enlarged cell bodies and unclear architecture, and the presence of damaged motor neurons with nucleus pyknosis and congestion of the blood vessels. The spinal cords of the tSCII group showed almost normal cell morphology, with reference to the control and shamoperated groups.

2.5. BM-MSC transplantation reduced apoptosis of nerve cells the spinal cords of rats with SCII Apoptosis in nerve cells was detected by TUNEL staining (Fig. 3B). The percentage of neural cells that underwent apoptosis in the control (5.7671.49%) and sham-operated (6.7671.84%) groups was less than those in the uSCII model group (48.3578.37%) and tSCII group (15.1074.14%). In the uSCII model group, numerous neural cells were strongly positive for TUNEL staining. However, the percentage of apoptotic neurons in the tSCII group was significantly lower than that of the uSCII model group (Po0.05).

2.6. BM-MSC transplantation reduced the level of CASP3 and increased the expression of NF-H in the injured spinal cord The expression of CASP3 was examined via immunohistochemistry. The level of CASP3 in the uSCII model group were significantly higher than that of either the control or

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Fig. 3 – Histopathological examination, detection of apoptotic cells and expressions of CASP3 and NF-H. (A) Histopathological examination of the spinal cord by H&E staining. The appearance of nerve cells was normal in the control and sham-operated ) with nuclear pyknosis and obvious blood vessel congestion ( ) were observed in the groups. Some damaged nerve cells ( spinal cords of the uSCII model group. The histopathological changes of the spinal cords of rats in the cell transplantation (tSCII) group were minimal and cell morphology was almost normal. (B) Detection of apoptotic cells in the spinal cord ), visualized by TUNEL staining. Numerous nerve cells were strongly positive for TUNEL staining in the uSCII model group ( but fewer in the control, sham-operated, and BM-MSC transplantation groups. (C) Expression of CASP3 in the spinal cord ) in the uSCII model group was significantly higher than visualized by immunohistochemistry. The expression of CASP3 ( that of the control, sham-operated, and cell transplantation groups (Po0.05). (D) Expression of NF-H in the spinal cord detected ) in the uSCII model group was significantly lower than that of the by immunohistochemistry. The expression of NF-H ( control, sham-operated, and tSCII groups (Po0.05). Scale bar¼20 μm. sham-operated groups, by 6.25- and 6.09-fold, respectively (Po0.05; Table 1; Fig. 3C). However, the expression of CASP3 in the tSCII group was 14.57% that of the uSCII model group (Po0.05). NF-H levels in the uSCII model group were significantly lower than those of the control, sham-operated, and tSCII groups (Po0.05; Fig. 3D). The level of NF-H in the tSCII group was 174% that of uSCII model group (Po0.05).

2.7. Transplantation of BM-MSCs significantly reduced the ratio of BAX to BCL2 The expressions of BAX and BCL2 were examined by double immunofluorescence and were observed in neural cells of all groups (Table 1, Fig. 4). BAX expression in the uSCII model group

was 146% that of the cell transplantation group, BCL2 expression was 74% that of the cell transplantation group. The ratio of BAX to BCL2 in the spinal cords of the BM-MSC transplantation group was 50% that of the uSCII group (Po0.05). No significant difference in the BAX to BCL2 ratio was found between the cell transplantation and control groups, or between the cell transplantation and sham-operated groups, indicating that the ratio in the cell transplantation group was approximately normal.

2.8. CM-Dil-labeled BM-MSCs migrated to the damaged spinal cord and differentiated into neurons and astrocytes Seven days after reperfusion, CM-Dil-labeled BM-MSCs were found in the damaged spinal cord of the recipient (MSC-SCII)

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Table 1 – The exprexssions of CASP3, NF-H, BCL2, and BAX in the spinal cord by immunohistochemistry and immunofluorescence (integrated optical density; mean7SD). Groups

CASP3

NF-H

BAX

BCL2

BAX:BCL2

Control Sham-operated uSCII model tSCII

315.750764.672 322.875753.550 2288.750783.570a,b 333.375736.730c

189.293758.842 166.604715.304 69.533712.798ab 120.982725.905abc

91.44874.246 105.17878.961 146.438713.099a,b 100.140712.893c

102.864713.938 101.13676.195 80.235712.328a,b 108.18678.073c

0.90470.139 1.04070.071 1.86370.340a,b 0.92570.095c

a b c

Po0.05 compared with the control group. Po0.05 compared with the sham-operated group. Po0.05 compared with the uSCII model group.

Fig. 4 – Expressions of BAX and BCL2 in the spinal cord using double labeling immunofluorescence. The expression of BAX was greatly increased in the uSCII model group while the expression of BCL2 was significantly declined in the uSCII model group when compared with that of the control and sham-operated groups. Transplantation of BM-MSCs appeared to effect a significant reduction in the ratio of BAX to BCL2 compared with the uSCII model group (Po0.05). Green: BAX; Red: BCL2; Blue nuclei: Hoechst 33342 staining. (A) control; (B) sham-operated; (C) uSCII model; and (D) tSCII. Scale bar¼ 10 μm.

rats, indicating homing to the damaged spinal cord and successful engraftment occurred. Results of the immunofluorescence staining showed the migrated BM-MSCs expressed microtubule-associated protein 2 (MAP2), a neuronal marker, and glial fibrillary acidic protein (GFAP), an astrocyte marker, suggesting BM-MSCs differentiated into neurons and astrocytes (Fig. 5).

3.

Discussion

In this study, we verified expressed the mesenchymal stem cells (MSCs) markers CD44 and CD90 but not CD45 by flow cytometry that BMSCs, indicating MSCs were not hematopoietic. Moreover, we found that neuronal apoptosis, expression of CASP3, and the ratio of BAX to BCL2 in the spinal cords

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Fig. 5 – CM-Dil-labeled BM-MSCs (red fluorescence) differentiated into neurons and astrocytes in the damaged spinal cord of the recipient (tSCII) rats at 7 days after reperfusion. (A) CM-Dil-labeled BM-MSCs (red) differentiated into astrocyte, expressing ). (B) CM-Dil-labeled BM-MSCs (red) differentiated into neuron, expressing neuron marker, astrocyte marker, GFAP (green) ( ). Blue nuclei were stained by Hoechst 33342. MAP2 (green) ( of SCII-induced rats transplanted with BM-MSCs were significantly lower than those of untreated (uSCII) rats. Transplantation of BM-MSCs by retro-orbital intravenous injection significantly improved the hind-limb motor functions in rats with SCII at the early phase of post spinal cord injury, and CM-Dil-labeled BM-MSCs was present and differentiated into neurons and astrocytes in the injured spinal cord. Our results indicated that BM-MSCs in rats with SCII had the ability to induce fast recovery, which might be due to the anti-apoptotic effect of BM-MSCs. It has been hypothesized that apoptosis in glia may result in inflammatory secondary injury (Colak et al., 2005; Shuman et al., 1997) and that apoptosis in neurons could have a dramatic negative effect on neurological functions (Li et al., 2000). These findings support the results of a previous study showing that BMMSCs decreased amyloid β peptide-induced apoptosis of primary cultured hippocampal neurons (Lee et al., 2010). They also reported that BM-MSCs had an anti-apoptotic effect in an acutely induced (with intra-hippocampal injection of amyloid β) mouse model of Alzheimer's disease. Additionally, Mackey et al. used TUNEL staining, H&E staining, electron microscopy, and DNA agarose gel electrophoresis to demonstrate that ischemia induced neuronal apoptosis in a rabbit model of spinal cord ischemia (Mackey et al., 1997). Apoptosis occurred in the affected neurons, as shown by positive TUNEL staining, apoptotic bodies, and nuclear condensation, and by the results of agarose gel electrophoresis, which revealed that apoptosis of neurons was a mechanism underlying delayed neuronal death. Hayashi et al. found that approximately 50% of motor neurons were TUNEL-positive, 2 days after 15 min of transient ischemia in a rabbit model of balloon catheterinduced spinal cord ischemia (Hayashi et al., 1998). In the present study, results of decreased expression of NF-H (also known as NEFH) confirmed axon loss in the injured part

which may be related to the neurologic deficit. Transplantation of BMSCs increased the expression of NF-H, which may be associated with improvement of the hindlimb motor functions. The most abundant cytoskeletal proteins are neurofilaments in large myelinated axons (Perrot et al., 2008), and NF-H is a neurofilament that is important in the stabilization of mature axons (Yabe et al., 2001). To investigate mechanisms underlying suppression of apoptosis after transplantation of BM-MSCs after SCII, in the present study we examined expressions of BCL2 and BAX in apoptosis, and demonstrated that the expression of BAX was greatly increased in the uSCII model group compared with that in the sham-operated group, while transplantation of BM-MSCs reduced the expression of BAX and the ratio of BAX to BCL2. However, there was no significant difference in BCL2 expression among the tSCII model group, the control and sham-operated groups. Moreover, the expression of CASP3 in the uSCII model group was significantly increased when compared to that in the sham-operated and tSCII model groups (Po0.05). Therefore, based on our studies, we assume that up-regulation of BAX, down-regulation of BCL2, and consequently the higher ratio of BAX/BCL2 may be directly associated with the release of cytochrome C and the higher expression of CASP3, and the elevated CASP3 finally resulted in apoptosis after SCII. Moreover, the BM-MSCs antagonized apoptosis by inhibiting up-regulation of BAX, down-regulation of BCL2 expression, and a decrease in the ratio of BAX to BCL2, resulting in suppression of CASP3 expression, and therefore a decrease in the number of apoptotic cells in the tSCII model group. Additionally, laser scanning confocal microscopic observation of the CM-Dillabeled BM-MSCs showed that CM-Dil was evenly distributed in the cytoplasm, a sign of homing of the BM-MSCs in the injured spinal cord. Theoretically, it is possible that the label

brain research 1561 (2014) 1–10

may leak from the labeled cells, particularly with cell death. However, if host cells engulf the transplanted cells, CM-Dil should not distribute in the cytoplasm evenly. Markus et al. (1997) found that after selective intraportal transplantation of hepatocytes labeled by Dil, only fluorescent debris was observed in spleen and pulmonary tissue after various organs were examined (Markus et al., 1997). The double immunofluorescence labeling showed that the CM-Dil-labeled BMMSCs expressed the neuronal marker, MAP2 and the astrocyte marker, GFAP, indicating homing and differentiation of the transplanted BM-MSCs into neurons and astrocytes in the injured spinal cord. This result suggested another potential mechanism underlying neural repair, which should be investigated further. In this study, the results showed that BM-MSCs could migrate into injured spinal cord tissue and repair SCII by an anti-apoptotic effect. While we observed that BM-MSCs could migrate successfully into the damaged spinal cord and repair SCII by reducing apoptosis, this repair effect was only observed until after seven days post-reperfusion. Although the long-term effect of BM-MSCs and the determination of the apoptotic pathways that are involved require verification, we have laid sufficient foundation for further study of SCII treatments with BM-MSCs, and we will provide a possible therapeutic approach for the management of SCII in the near future.

4.

Experimental procedures

4.1.

Rats

Adult and 5–7 day-old Sprague–Dawley rats (SD rats) were obtained from the Center of Laboratory Animals at Jilin University (Changchun, PR China). All procedures were performed in accordance with the guidelines of the Animal Care and Use Committee of Jilin Province of China. Animals were fed standard rat food and water and housed under controlled temperature with 12 h light and 12 h dark cycle.

4.2.

Culture of rat BM-MSCs

Cells from the bone marrows of 5–7 day-old SD rats (n ¼5) were cultured as reported previously with slight modifications (Guo et al., 2005). In brief, five SD rats were euthanized. They were then sterilized with 75% ethanol and the tibias and femurs of the rats were dissected. The bone marrow was flushed with a medium consisting of Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (DMEM/F12, Gibco, Grand Island, NY), penicillin (100 U/mL) and streptomycin (100 U/mL; Sigma, St. Louis, USA). The bone marrow cells flushed with the medium were gently drawn up and down with a 3-cc syringe and 21-gauge needle, and then a single cell suspension was obtained. After the bone marrow was centrifuged and collected, cells were adjusted to 1  106 cells/mL and seeded in 75-mL cell flasks containing DMEM/F12 supplemented with 10% bovine fetal calf serum (Hyclone, Logan, USA), penicillin, and streptomycin and incubated at 37 1C with 5% CO2 in a humidified incubator. Half of the culture medium was refreshed

7

72 h after seeding. Seven days later, when cells were confluent, the culture was passaged with trypsin.

4.3.

Cultured BMSC biomarker analysis

The third-passage BMSCs were analyzed for mouse anti-rat marker mouse monoclonal antibody CD44 (eBioscience, San Biego, CA, USA), CD45 and CD90 (BioLegend, San Biego, CA, USA). Briefly, 5  105 BMSCs in 100 μL PBS were incubated with the antibodies at room temperature for 45 min in the dark, then the cells were washed three times with PBS and resuspended in PBS, whereupon flow cytometry was performed.

4.4. BM-MSCs treated with CM-Dil cell-labeling solution and efficacy of CM-Dil labeling of BM-MSCs To label the BM-MSCs, CM-Dil was chosen at the concentration of 4 μg/mL. A stock solution of CM-Dil (Molecular Probes, Invitrogen, Carlsbad, California, USA) was prepared at 1 mg/ mL in dimethyl sulfoxide (Sigma, St. Louis, USA) in accordance with the manufacturer's instructions. Before transplantation, BM-MSCs were digested with trypsin, centrifuged, and the collected cell pellets were stained with 4 μg/mL CMDil for 15 min on ice. After labeling, cells were washed with phosphate-buffered saline (PBS) and re-suspended in fresh DMEM/F12. The labeling rate of BM-MSCs was determined by flow cytometry assay.

4.5.

Preparation of the SCII model

Spinal cord ischemia was induced using the method described by Zivin and DeGirolami (1980) with a few modifications. Briefly, SD rats (body weight: 220720 g) were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL/kg). The rats’ body temperature was maintained at 3770.5 1C (measured rectally) with a heating unit. An abdominal incision (4 cm) was made, and the abdominal aorta was separated at the level of the renal artery and occluded 0.5 cm below the left renal artery with an aneurysm clip for 60 min. Then the clip was removed, and the incision was sutured. All the animals survived this procedure.

4.6.

Experimental design

Forty Sprague–Dawley rats (20 male) were equally divided into 4 groups, with an equal number of males and females: the blank control (non-operated, non-treated), shamoperated (non-treated), untreated SCII (uSCII, the same volume saline solution injected as the transplanted group), and BM-MSC-transplanted SCII (tSCII). The same procedure was performed on the sham-operated rats as those in the uSCII model group but without occlusion of the aorta. For the tSCII group, transplantation of BM-MSCs was performed by retro-orbital intravenous injection of CM-Dil-labeled BMMSCs (5  106) at 1 h and 24 h after the reperfusion.

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4.7.

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Transplantation of CM-Dil-labeled BM-MSCs

Ten rats in the tSCII group were anesthetized with ethyl ether. One hour after the reperfusion, BM-MSCs (5  106, in 0.1 mL) were administered by retro-orbital injection using a r27.5-gauge 0.5-in insulin needle and syringe. The right eyeball of the rats was partially protruded from the eye socket by applying gentle pressure to the skin around the eye. Then the needle was beveled down at an angle of approximately 30 degree and inserted into the retro-orbital sinus at the medial canthus, following the edge of the eyeball down, until the needle tip was at the base of the eye. The cells were injected slowly and smoothly. The needle was then slowly and smoothly withdrawn. After the injection was completed, each rat was put back into its cage for recovery. 24 h after reperfusion, the injection of BM-MSCs was repeated as above, except into the retro-orbital sinus at the medial canthus of the left eyeball.

4.8.

Evaluation of hind-limb motor function

Hind-limb motor function was scored daily by the Basso– Beattie–Bresnahan (BBB) locomotor rating scale during the first week after reperfusion (Barros Filho and Molina, 2008), one time a day. Briefly, the BBB scale includes monitoring the movement of the hindlimbs and joints, weight-bearing ability, forelimb-hind-limb coordination, paw position, and tail elevation with scores ranging from 0 (can not observe movement of the hindlimbs) to 21 (normal movement) according to different degrees of changes in the above aspects.

4.9.

Histopathology of excised spinal cords

L3–L4 segments of the spinal cords obtained seven days after reperfusion were fixed with 10% neutral buffered formalin and post-fixed in paraffin blocks for later sectioning and hematoxylin and eosin (H&E) staining.

4.10. Detection of CM-Dil-labeled BM-MSCs with neuronal and astrocyte markers in injured spinal cords The spinal cord tissues were dissected 7 days after reperfusion, fixed with 10% neutral buffered formalin for one day, and precipitated with 25% sucrose solution for one day. Frozen sections were then prepared and stained with immunofluorescence for MAP2 and GFAP. Transplanted CM-Dillabeled BM-MSCs stained with MAP2 and GFAP were detected using a laser scanning confocal microscope.

4.11.

Detection of apoptosis in excised spinal cords

TdT-mediated dUTP nick end Labeling (TUNEL) staining was performed using an In Situ Cell Death Detection Kit (POD, Roche, Basel, Switzerland) in accordance with the manufacturer's recommendations. Briefly, dewaxed, dehydrated, 5-μm sections of excised spinal cord specimens were treated with protease K (20 μg/mL, Roche, Basel, Switzerland) and incubated at 37 1C for 20 min. Endogenous peroxidase activity was blocked using 3% H2O2 in methanol. The sections were incubated with the TUNEL-reaction mixture at 37 1C for

60 min. Then these slides were incubated with ConverterPOD at 37 1C for 30 min. Finally, the slides were developed using a 3,3’-diaminobenzidine (DAB) Substrate Kit (Fuzhou Maixin Biotechnology, Fu Zhou, PR China). Hematoxylin was used as a nuclear counterstaining reagent. The number and percentage of TUNEL-positive cells in each section were analyzed using image analysis software (Image-pro plus 6.0). The grey matter of the spinal cord was observed, 5 sections per animal, 5 fields per section. All the cells in all the chosen fields were used for quantitative analysis. The measurement frame was 640  640 pixels, equal to an area 500  500 μm2. Each measured area encompassed approximately 200 cells.

4.12. Immunohistochemistry and double immunofluorescence labeling of excised spinal cords Immunohistochemical staining was performed in accordance with the manufacturer's instructions (UltraSensitive TM S-P kit with DAB as a chromogen; Fuzhou Maixin Biotechnology, Fu Zhou, PR China). Sections from formalin-fixed, paraffinembedded spinal cord tissues were dewaxed, rehydrated, and retrieval of antigens was performed. After incubation with 3% H2O2 in methanol, and then normal non-immune goat serum, the sections were incubated with rabbit anti-active CASP3 polyclonal antibody at a dilution of 1:200 (Abcam, Cambridge, UK) and rabbit polyclonal antibody neurofilament-H (NF-H) (1:200 dilution; ProteinTech, Chicago, USA) at 4 1C, followed by biotinylated goat anti-rabbit IgG (Fuzhou Maixin Biotechnology, Fu Zhou, PR China) for 20 min at room temperature, and subsequently incubated with streptavidin–peroxidase (Fuzhou Maixin Biotechnology, Fu Zhou, PR China). PBS replaced primary antibody as the negative control. DAB was applied for visualization of peroxidase activity. Finally, the sections were counterstained with hematoxylin. For immunofluorescence labeling, the paraffin-embedded sections were dewaxed, rehydrated, and retrieval of antigens was performed as described above. The paraffin-embedded and frozen sections were then treated with 1% TritonX-100 in PBS, washed, incubated with normal non-immune goat serum, washed, and incubated with primary antibodies at 4 1C overnight. Primary antibodies included BCL2 (1:200, rabbit polyclonal antibody) and BAX (1:200, mouse monoclonal antibody), both from Santa Cruz Biotechnology, Santa Cruz, CA, USA, MAP2 (1:200, rabbit polyclonal antibody, Proteintech Group, Chicago, USA) and GFAP (1:400, mouse monoclonal antibody, Proteintech Group, Chicago, USA). After washing with PBS, secondary antibodies were applied at room temperature for 40 min. The secondary antibodies (Molecular Probes, Eugene, OR, USA) were Alexa Fluor goat anti-mouse 488 at 1:400 for mouse primary antibodies, Alexa Fluor goat anti-rabbit 594 and Alexa Fluor goat anti-rabbit 488 at 1:400 for primary rabbit antibodies. The coverslips were then washed and stained with Hoechst 33342 (Sigma, St. Louis, USA). Sections were observed under a laser scanning confocal microscope, and images were collected. Three sections per rat and 5 fields per section were selected for analysis. The integrated optical density of the entire grey matter of the L3–L4 segments was measured using the image analysis software (Image-pro plus 6.0).

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4.13.

Statistical analyses

Data are presented as mean7standard deviation. Comparisons among the four groups were performed using one-way analysis of variance, and subsequently analyzed with Fisher's least significant difference test using the SPSS 13.0 software (SPSS, Chicago, USA).

Conflict of interest We declare we have no conflict of interest.

Acknowledgments Project supported by the Foundation of Science and Technology Development Program of Jilin Provincial Science and Technology Department, PR China (Grant no. 200905183), Scientific Research Foundation of Jilin Department of Health of China (Grant no. 20082041), Creative Program of Scientific Frontiers and Cross-disciplinary of Jilin University Basic scientific research, PR China (Grant no. 200903114).

r e f e r e n c e s

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