A combination of taxol infusion and human umbilical cord mesenchymal stem cells transplantation for the treatment of rat spinal cord injury

A combination of taxol infusion and human umbilical cord mesenchymal stem cells transplantation for the treatment of rat spinal cord injury

brain research 1481 (2012) 79–89 Available online at www.sciencedirect.com www.elsevier.com/locate/brainres Research Report A combination of taxol...

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brain research 1481 (2012) 79–89

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

A combination of taxol infusion and human umbilical cord mesenchymal stem cells transplantation for the treatment of rat spinal cord injury Zhou Zhilaia, Zhang Huia, Jin Anmina, Min Shaoxionga, Yu Boa, Chen Yinhaib,n a

Department of Orthopedics, Zhujiang Hospital, Southern Medical University, 253 Gongye road, 510282 Guangzhou, China. Department of Rehabilitation, Zhujiang Hospital, Southern Medical University, 253 Gongye road, 510282 Guangzhou, China

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art i cle i nfo

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Article history:

Background and purpose: Studies have shown that the administration of Taxol, an anti-

Accepted 27 August 2012

cancer drug, inhibited scar formation, promoted axonal elongation and improved loco-

Available online 31 August 2012

motor recovery in rats after spinal cord injury (SCI). We hypothesized that combining Taxol

Keywords:

with another promising therapy, transplantation of human umbilical mesenchymal stem

Spinal cord injury

cells (hUCMSCs), might further improve the degree of locomotor recovery. The present

Taxol

study examined whether Taxol combined with transplantation of hUCMSCs would produce

Umbilical cord

synergistic effects on recovery and which mechanisms were involved in the effect.

Mesenchymal stem cells Transplantation

Methods: A total of 32 rats subjected to SCI procedures were assigned to one of the following four treatment groups: phosphate-buffered saline (PBS, control), hUCMSCs, Taxol, or TaxolþhUCMSCs. Immediately after injury, hUCMSCs were transplanted into the injury site and Taxol was administered intrathecally for 4 weeks. Locomotor recovery was evaluated using the Basso, Beattie and Bresnahan locomotor (BBB) rating scale. Survival of the transplanted human cells and the host glial reaction in the injured spinal cord were studied by immunohistochemistry. Results: Treatment with Taxol, hUCMSCs or TaxolþhUCMSCs reduced the extent of astrocytic activation, increased axonal preservation and decreased the number of caspase3þ and ED-1þ cells, but these effects were more pronounced in the TaxolþhUCMSCs group. Behavioral analyses showed that rats in the TaxolþhUCMSCs group showed better motor performance than rats treated with hUCMSCs or Taxol only. Conclusions: The combination of Taxol and hUCMSCs produced beneficial effects in rats with regard to functional recovery following SCI through the enhancement of antiinflammatory, anti-astrogliosis, anti-apoptotic and axonal preservation effects. & 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Traumatic spinal cord injury (SCI) induces neural cell death, axonal degeneration, and destruction of the microvasculature. n

Corresponding author. Fax: þ86 20 61643218. E-mail address: [email protected] (C. Yinhai).

0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.08.051

These events trigger a subsequent cascade of pathological changes (so-called secondary events), including freeradical release, hyperplasia of astrocytes, calcium-mediated damage, hemorrhagic necrosis, mitochondrial dysfunction,

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and inflammatory responses, that lead to delayed cellular dysfunction and death (Guest et al., 2005; Waxman et al., 1991). Moreover, the poor trophic support and growthinhibitory nature of the environment of the adult central nervous system (CNS) is hostile to endogenous spinal cord regeneration. Intensive efforts have been made to develop therapeutic strategies to minimize the extent of neurological disabilities and to promote the recovery of function after SCI. However, no single neuroprotective agent has been approved for clinical use by the US Food and Drug Administration, underlining the need to focus on strategies that simultaneously affect multiple injury mechanisms. In this context, we hypothesized that a combination of therapeutic strategies might be more effective than a single strategy for promoting functional recovery after SCI. Taxol is a clinically approved anti-cancer drug. Taxol stabilizes microtubules and, as a result, interferes with the normal breakdown of microtubules during cell division (Vyas and Kadow, 1995). Taxol can prevent the formation of axon retraction bulbs and decrease axonal degeneration after SCI and can overcome myelin inhibition of neurite outgrowth in vitro (Erturk et al., 2007). Taxol also exhibits immunomodulatory action on immune cells, such as microglia and macrophages. For example, Taxol can reduce the infiltration/activation of ED-1þ cells (macrophages/microglia) after optic nerve injury (Sengottuvel et al., 2011). A recent study also indicated that moderate microtubule stabilization by Taxol decreased scar formation and prevented accumulation of chondroitin sulfate proteoglycans (CSPGs) in an experimental model of rat SCI (Hellal et al., 2011). Cell transplantation is another promising strategy for the treatment of SCI. The grafted cells could provide trophic support for neurons and manipulate the environment within the damaged spinal cord to facilitate axon regeneration or to promote plasticity in the lesioned spinal cord. Various types of multipotent stem cells, including embryonic stem cells (ES) (Deshpande et al., 2006), neural stem cells (NSC) (Yasuda et al., 2011) and mesenchymal stem cells (MSC) (Ide et al., 2010) are currently under investigation as potential alternative cell sources for cell transplantation. Theoretically, ES cells are the best candidates for treating SCI; however, their potential is limited by ethical issues. The number of NSCs decreases in neurogenesis, and NSCs undergo replicative senescence over time. In this context, mesenchymal stem cells (MSC) are promising candidates as cumulative evidence shows both the multipotency of MSCs and their capability to exert a neuroprotective effect after CNS injury through the paracrine production of mitogenic, antiapoptotic, and trophic factors (Nakajima et al., 2012; Sakai et al., 2011; Schira et al., 2011). Among the MSCs, human umbilical mesenchymal stem cells (hUCMSCs) appear to have several advantages. They offer a noncontroversial, readily available source of cells and can be obtained through a lowcost, noninvasive collection method (Yang et al., 2012). Furthermore, hUCMSCs are isolated from fetal structures during the perinatal period and are better tolerated following transplantation, resulting in a lower incidence of graft versus host disease compared with other types of postnatal MSCs (Cho et al., 2008).

The treatment of SCI requires a multifaceted strategy because of the multiple potential mechanisms that hinder spinal cord recovery (Kim et al., 2007). The aim of this study is to evaluate whether a therapeutic combination of hUCMSCs and Taxol enhances the beneficial effects of treatment.

2.

Results

2.1.

Characterization of hUCMSCs

The isolated hUCMSCs demonstrated a fibroblast-like morphology in confluent layers in culture. In agreement with previous observations (Novikova et al., 2011), all of the hUCMSCs were immunopositive for vimentin, laminin and fibronectin. The percentage of hUCMSCs expressing Nestin and Ki67 was 9.371.4% and 56.6575.35%, respectively. The hUCMSCs did not express CD34, indicating that they were of non-hematopoietic origin (Fig. 1).

2.2.

Cell survival and migration in vivo

SCI was induced in female Sprague-Dawley rats by dropping a 10 g metal rod from a height of 7.5 cm using an NYU impactor. Immediately after injury, 2  105 hUCMSCs were transplanted at a distance of 2 mm rostral and 2 mm caudal to the site of injury, respectively. The survival and migration of hUCMSCs in the hUCMSCs group and in the hUCMSCsþ Taxol group was assessed 1 week after transplantation in harvested sagittal tissue sections. Grafted human stem cells were clearly detected by immunohistochemical staining with a specific anti-human nuclei antibody (hNu, MAB1281). One week after transplantation, we found extensive survival of the human cells (Fig. 2A). The percentage of surviving cells versus total transplanted cells was 37.8710.5% and 32.579.2% for the hUCMSCs group and the hUCMSCsþTaxol group, respectively (p40.05, N ¼ 4 for each group) (Fig. 2B). The majority of the surviving cells were observed at the epicenter of the lesion, suggesting that the grafted cells migrated toward the injury sites. We investigated whether the human stem cells exhibited active cell division/proliferation after transplantation by double-labeling for hNu and Ki67. Most of the grafted cells did not express Ki67 (3.171.6% and 2.571.2% for the hUCMSCs group and the hUCMSCsþTaxol group, respectively, p40.05, N¼ 4 for each group) (Fig. 2C), indicating that they ceased proliferation after transplantation into the injured spinal cord. Ki67-positive cells were randomly dispersed across the graft area without evidence of clustering in specific sites.

2.3.

Fate of transplanted cells

Histological examination revealed that the hUCMSCs survived for 4 weeks after transplantation. To evaluate the neural differentiation potential of the hUCMSCs in the spinal cord environment, the expression of neuronal and glial marker proteins was analyzed using immunohistochemistry. The results showed that, whether they were with within or outside of the lesion zone, the hUCMSCs failed to express the

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Fig. 1 – Characterization of the hUCMSCs used for transplantation studies. hUCMSCs immunostained for laminin (A), fibronectin (B), and vimentin (C). The percentage of hUCMSCs expressing Nestin (D) and Ki67 (E) was 9.371.4% and 56.6575.35%, respectively; hUCMSCs were negative for CD34 (F). Scale bar ¼50 lm.

Fig. 2 – Transplantation of hUCMSCs into the injured rat spinal cord. (A) Detection of transplanted hUCMSCs by specific antihuman nuclei antibody (hNu) staining 1 week after transplantation. Extensive human cell survival in the hUCMSCs group (A–D) and the hUCMSCsþTaxol group (E–H). hNuþ, green (C, G) was rarely associated with the cell cycle marker Ki67, red (B, F). DAPI counterstain, blue (A, E). Arrows indicate a double-labeled cell. (I) Quantitative analysis of the hNuþ cell numbers in the hUCMSCs and hUCMSCsþTaxol groups. (J) Quantitative analysis of the hNuþ/Ki67þ cell numbers in the hUCMSCs and hUCMSCsþTaxol groups. Scale bar¼ 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

early neuronal cell marker bIII-tubulin or the glial proteins GFAP and GNPase. Therefore, the hUCMSCs did not differentiate into neurons, astrocytes or oligodendrocytes after transplantation into the acutely injured spinal cord (Fig. 3).

2.4.

Microglial/macrophage infiltration and apoptosis

Histological quantification of the inflammatory infiltrations and apoptotic cells were undertaken to confirm the anti-

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Fig. 3 – hUCMSCs remain undifferentiated 4 weeks following grafting into the injured spinal cord. Double fluorescence immunohistochemistry revealed that the grafted hUCMSCs were negative for an early neuron marker (bIII-Tubulin), an astrocyte marker (GFAP) and an oligodendrocyte marker (CNPase). Scale bar ¼50 lm.

Fig. 4 – Histological analysis of inflammation and apoptosis. Representative images for ED-1 and caspase-3 immunohistochemistry in sagittal sections from SCI animals treated with PBS, Taxol, hUCMSCs and hUCMSCsþTaxol 7 days post-injury. hUCMSCsþTaxol treatment enhanced the anti-inflammatory and anti-apoptotic effects afforded by the isolated therapies (Po0.05 ANOVA-Bonferroni, compared to control or other groups#). Scale bar¼ 50 lm.

inflammatory effects of hUCMSCsþTaxol at 7 days postinjury (Fig. 4A). Initially, we observed intergroup differences in the inflammatory/apoptotic cell counts, including ED-1 and caspase-3 (Po0.05, ANOVA-Bonferroni), among the four treatment groups. We further analyzed the values using intergroup comparisons. It was found that the hUCMSCsþ Taxol group possessed significantly fewer areas of ED-1þ cells

than the control group (Po0.05, ANOVA-Bonferroni). The hUCMSCs group and the Taxol group also showed a reduction in ED-1þ area versus the control group (all po0.05), but this was less pronounced than in the hUCMSCsþTaxol group (Fig. 4A and B) (control: 0.24670.038/mm2, Taxol: 0.1377 0.033/mm2, hUCMSCs: 0.15270.045/mm2, hUCMSCsþTaxol: 0.04670.017/mm2).

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An analysis of active caspase-3þ apoptotic cell numbers showed that the hUCMSCsþTaxol and hUCMSCs groups had lower counts than the control group (all Po0.05) and that the hUCMSCsþTaxol group had lower counts than the hUCMSCs group (Po0.05). However, the Taxol group showed no reduction in caspase-3þ apoptotic cell numbers (Fig. 4A and C) (control: 38.675.3 cells/field, Taxol: 33.277.3 cells/field, hUCMSCs: 15.774.8 cells/field, hUCMSCsþTaxol: 7.871.6 cells/field).

2.5.

Gliosis

Gliosis was quantified by measuring the density of GFAP immunoreactivity. In the spinal cord tissue sections of control animals, dense GFAP immunoreactivity was detected surrounding the lesion sites at 4 weeks post-injury, and these astrocytes were packed tightly together as a scar barrier. In contrast, astrocytic fronts were less prominent and GFAP immunoreactivity in the adjacent regions was much weaker in the hUCMSCsþTaxol group than in the other 3 groups. Quantification showed that the GFAP staining intensities were significantly reduced by treatment with hUCMSCs and Taxol (all Po0.05) and that the extent of the decrease caused by treatment with hUCMSCsþTaxol was significantly larger than that of hUCMSCs or Taxol alone (Fig. 5).

2.6.

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Axonal staining

To examine whether treatment with hUCMSCsþTaxol affected the preservation of neurofilaments, we performed immunohistochemical analysis with an anti-neurofilament H (NF-200) mAb 4 weeks post-injury (Fig. 6A–D, I). Compared to the control group, the hUCMSCs group and the Taxol group exhibited greater preservation of NF-200þ axons at the rostral, caudal, and center segments of the injury. The maximum preservation of NF-200þ axons was observed in the hUCMSCsþTaxol group. The descending serotonergic raphespinal axons are critical for the recovery of hind limb locomotor function in rat SCI. We therefore evaluated whether the staining of these axons was enhanced in the caudal of the lesion in the hUCMSCsþTaxol group. The serotonergic raphespinal axons were immunohistochemically analyzed by a mAb that specifically reacts with 5-hydroxytryptamine (5-HT), which is synthesized within the brainstem. On the caudal side of the lesion site, the area of 5HTþ fibers was significantly greater in the hUCMSCsþTaxol group than in the other 3 groups (control: 0.0270.002/mm2, Taxol: 0.0470.009/mm2, hUCMSCs: 0.03870.001/mm2, hUCMSCs þTaxol: 0.13570.019/mm2) (Fig. 6E–H, J).

2.7.

Cavitation analysis

A quantitative analysis of the volume of the cavitations formed in all the groups was performed 4 weeks after SCI. The measurements of total lesion volume revealed the most significant reduction in the hUCMSCsþTaxol group (control: 33.5875.2%, Taxol: 29.874.2%, hUCMSCs: 21.672.9%, hUCMSCsþTaxol: 15.8þ4.8%, Po0.05 between hUCMSCsþTaxol and control). The hUCMSCs group also showed a reduction in lesion volume versus the control group (Po0.05), but this was less pronounced than in the hUCMSCsþTaxol group (Po0.05 versus hUCMSCs). However, the Taxol group showed no significant reduction in lesion volume (P40.05 versus control) (Fig. 7A).

2.8.

BBB score

As shown in Fig. 7B, all of the animals displayed complete hind limb paralysis after SCI. In the following days, locomotor performance substantially improved. Beginning in the 2nd week, the BBB scores in the treated groups (hUCMSCsþTaxol, hUCMSCs, and Taxol) were significantly higher than in the control group. The animals in the hUCMSCs and Taxol groups showed a similar recovery over time. Four weeks post-transplantation, the highest functional recovery was observed in the hUCMSCsþTaxol group in comparison with the other 3 groups (po0.05, ANOVA-Bonferroni) (control: 8.770.3, Taxol: 11.370.5, hUCMSCs: 11.570.3, hUCMSCsþTaxol: 13.570.4). Fig. 5 – Histological analysis of glial scarring. Representative images of GFAP immunostained longitudinal sections from animals with PBS control (A), Taxol (B), hUCMSCs (C) and hUCMSCsþTaxol (D) treatment, sacrificed 4 weeks after transplantation. Dotted lines indicate the lesion borders. Scale bar ¼100 lm. (E) Quantification of GFAP intensities (# Po0.05 versus all other three groups,  Po0.05 versus the control group). Scale bar¼ 100 lm.

3.

Discussion

This study demonstrates that the combined therapeutic treatment of Taxol infusion with hUCMSCs transplantation has greater therapeutic effects than the use of Taxol or hUCMSCs alone following SCI. First, compared to the other

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Fig. 6 – Axonal staining in injured spinal cord 4 weeks post-injury. Immunoreactive staining (A–D) and quantification (I) of NF-200þ fibers in the epicenter of the lesion. Immunoreactive staining (E–H) and quantification (J) of 5-HTþ fibers at the lumbar intumescence. (# Po0.05 versus the other three groups,  Po0.05 versus the control group). Scale bar in (A–D)¼100 lm, scale bar in (E–H)¼ 50 lm.

groups, the rats in the hUCMSCsþTaxol group showed better functional recovery, as measured by BBB scores. It was found that significant functional recovery began 2 weeks post-injury and increased gradually. Second, morphological analysis using HE staining showed that the lesion cavities of the hUCMSCsþTaxol rats were smaller than those of the control, Taxol, or hUCMSCs rats. Finally, immunohistochemical analysis showed increased axonal preservation, as well as decreased inflammation, apoptosis and astrogliosis, in the injury site. hUCMSCs are considered a promising therapy for the treatment of SCI (Gong et al., 2012; Yang et al., 2008), hUCMSCs grafted into SCI models showed variable degrees of functional improvement in different established animal models. Yang, reported that the direct injection of 105 hUCMSCs into a complete SCI rat model significantly promoted functional recovery 3 weeks after the graft and continued for up to 16 weeks (Yang et al., 2008). Another study by Hu, demonstrated that the transplantation of 4  105 hUCMSCs 1 day after contusive SCI significantly promoted functional recovery 5–8 weeks after the graft (Hu et al., 2010). In the present study, hUCMSCs grafted alone also promoted the recovery of locomotor function, compared to the control group using a BBB score. These results suggest that hUCMSCs are active in different SCI models. However, the mechanisms underlying the beneficial effects of hUCMSCs are not fully

understood; they most likely do not involve transdifferentiation with cell replacement. As shown by our double immunofluorescence analysis, we found no evidence that hUCMSCs differentiated into neuronal or glial cells after transplantation into the injured rat spinal cord. Our results encourage and support the idea that the transplantation of hUCMSCs may exert therapeutic effects by modulating the release of growth factors and anti-inflammatory cytokines into the injury environment. Several soluble factors are directly released by hUCMSCs, including hepatocyte growth factor (HGF), brainderived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), nerve growth factor (NGF), vascular endothelial growth factor (VEGF) and anti-inflammatory cytokines (Arufe et al., 2011; Koh et al., 2008; Majore et al., 2011). Moreover, it has been shown that hUCMSCs can modulate the activity of microglia, inducing these cells to release growth factors and/or antiinflammatory cytokines (Liao et al., 2009; Zhang et al., 2011). In the present study, the hUCMSCs may have released soluble factors that contributed to a reduction in apoptosis and tissue preservation. To make the transplantation of hUCMSCs more effective, we combined hUCMSCs transplantation with infusion of Taxol. Why did we choose this particular combinatorial strategy? We know that axons in the CNS do not normally regrow after injury, whereas lesioned axons in the peripheral nervous system can regenerate. This phenomenon was partly

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Fig. 7 – Behavioral evaluation and cavitation analysis after spinal cord injury. (A) Multiple comparisons of cystic cavity formation among the four groups. The mean cystic cavity size was smallest in the hUCMSCsþTaxol group, compared to the other three groups (# Po0.05 versus the other three groups,  Po0.05 versus the control group). (B) Time course of the functional recovery of the hindlimbs after SCI. The combined treatment of Taxol and transplantation of hUCMSCs resulted in the greatest functional recovery. Data represent mean7SD (# Po0.05 versus the other three groups,  Po0.05 versus the control group).

explained by the presence of a number of myelin-derived inhibitors in the CNS. When these myelin-associated inhibitors bind to the NgR, the RhoA-ROCK pathway is activated and axon outgrowth is inhibited. However, overcoming inhibitory signaling alone is insufficient to enable long-distance axon regeneration(Zheng et al., 2005). Studies have shown that microtubules and dynamic rearrangements of microtubules are essential for axon outgrowth (Erturk et al., 2007; Williamson et al., 1996). Unfortunately, lesioned CNS axons form ‘‘retraction bulbs’’ at their proximal stumps that contain disorganized microtubules. It has been demonstrated that preventing microtubule disorganization by treatment with Taxol inhibits the formation of retraction bulbs and promotes axonal regeneration in the CNS. After Taxol was found to induce axonal elongation in the presence of myelin inhibitors, we wondered if a combination of Taxol infusion with hUCMSCs transplantation would further promote axonal regeneration in a rat model of SCI. Interestingly, we found that the combinatorial treatment of Taxol with hUCMSCs

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increased the area 5-HTþ fibers on the caudal side of the lesion, enhanced the preservation of neuronal fibers in the center of the lesion and significantly reduced mean cystic cavity size compared to the other groups. These histological results were consistent with a significant improvement in locomotor activity. Furthermore, our in vivo study demonstrated that the distribution and migration pattern of hUCMSCs was not altered between groups, suggesting that the Taxol concentrations we used were not toxic to the hUCMSCs and that the difference in functional outcome was not the result of a difference in cell survival or cell distribution. Treatment with hUCMSCsþTaxol reduced the number of ED-1þ cells in the first week after SCI and clearly improved the neuroprotection and functional recovery afforded by the treatment of SCI with hUCMSCs. Thus, the anti-inflammatory effect of hUCMSCsþTaxol may be of value in the treatment of SCI. There is substantial evidence suggesting that hUCMSCs may exert therapeutic effects by suppressing the immune response following CNS trauma, including SCI. Taxol is an anti-proliferative drug known to affect cell migration. A recent study reported that local Taxol treatment dramatically decreased the infiltration/activation of ED-1þ cells (macrophages/microglia) in the optic nerve after injury. The findings from our immunohistochemical analysis indicated that the combinatorial treatment of Taxol and hUCMSCs further attenuated the infiltration/activation of microglia and macrophages in the injured spinal cord. Another benefit of the use of Taxol with hUCMSCs for the treatment of SCI may be to reduce gliosis. Glial scars are thought to be a major impediment for axon regeneration following SCI. Hypertrophic astrocytes pack together tightly to form a scar barrier and express inhibitory molecules, such as CSPGs. Several studies have reported that the deletion of GFAP and the enzymatic degradation of CSPGs by chondroitinase ABC enhance axonal regeneration and improve functional recovery after SCI (Coumans et al., 2001; Jefferson et al., 2011; Tuszynski et al., 2003). A key event in glial scarring after SCI is the activation of transforming growth factor-b(TGF-b) signaling (Logan et al., 1994). Following SCI, TGF-bexpression dramatically increases and activates the TGF-b/Smad signaling pathway. Active TGF-b,via TGF-breceptor/Smad signaling in astrocytes, induces scar formation and upregulates neurocan, an inhibitory CSPG. Previous studies have demonstrated that microtubule destabilization modulates TGF-b signaling by inducing phosphorylation of Smads and translocation of microtubule bound-transcription factors from the cytoplasm to the nucleus (Dong et al., 2000; Sengottuvel and Fischer, 2011). Thus, microtubule stabilization by Taxol enhances the binding of Smad to microtubules, thereby diminishing the translocation of Smads to the nucleus and abrogating TGFb signaling. Consistent with these observations, the present study provides evidence that compared to the control group, Taxol infusion significantly reduced gliosis around the injury site 1 month after SCI. Furthermore, the reduced fibrotic scarring is more obvious in the hUCMSCsþTaxol group compared to the Taxol only group. It appears that transplantation of hUCMSCs in conjunction with Taxol infusion has a synergistic effect on suppressing astrocytic glial scar formation after SCI. Indeed, as we discussed previously, hUCMSCs can secrete a range of neurotrophic factors after

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transplantation into the injured spinal cord. Among the factors, HGF is one of the most powerful modulators of glial scarring. A recent study reported that the ex vivo delivery of HGF markedly diminished TGF-bisoform levels and attenuated astrocytic scar formation in an in vivo SCI model and increased the extent of axonal growth and recovery of locomotor function (Jeong et al., 2011). Perhaps combinatorial treatment with hUCMSCs and Taxol may reduce gliosis by suppressing the expression of TGF-b in the injured spinal cord. There were some limitations to this study. We conducted BBB testing to assess any functional benefits of the treatment. A major limitation of the BBB locomotor rating scale is the accurate assessment of forelimb–hindlimb coordination. For an accurate assessment of coordination, an observer must observe at least three, preferably four, paws of the rat at the same time (Basso et al., 1995; Koopmans et al., 2005). In practice, this is very difficult, even when two highly experienced observers are involved. In addition, the BBB score did not include an assessment of climbing. Thus, these limitations can have major implications for the final assessment of locomotor function in the present study. In conclusion, the combination of Taxol infusion and transplantation of hUCMSCs exhibited significant advantages over any single therapy alone. The effects of this treatment strategy were not simply the superposition of the two single therapies but the result of mutual benefits of combining the Taxol and hUCMSCs. The coordinated effects of Taxol and hUCMSCs on the reduction of microglia/macrophage infiltration and astrocytic scar formation may play critical roles in the promotion of spinal cord tissue repair and hindlimb recovery in rats with SCI.

4.

Experimental procedures

4.1.

Cell culture and identification

Ethical approval was obtained from Zhujiang hospital, Southern Medical University, China, and written informed consent was obtained from the umbilical cord donors. The isolation and culture of hUCMSCs were carried out as previously described (Shang et al., 2011). Briefly, fresh human umbilical cords were obtained after birth and processed within 4 h. The umbilical cord vessels were removed, and the remaining tissue was then diced into small fragments. The explants were transferred into 50 ml culture flasks containing Dulbecco’s modified essential media/nutrient mixture F12 (DMEM/F12, Gibco BRL, Grand Island, NY, USA), along with 10% fetal bovine serum (Gibco). The cultures were left undisturbed for 4–6 days to allow migration of cells from the explants, at which point the media was replaced. The cultures were maintained at 37 1C in an incubator containing 5% CO2 and were re-fed and passaged as necessary. To characterize the hUCMSCs in vitro, immunocytochemistry was performed. The cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X100 in PBS for 5 min. After blocking with 10% goat serum for 1 h, the cells were incubated with the following primary antibodies: mouse anti-vimentin (1:200; Sigma, St Louis,

MO), mouse anti-fibronectin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-laminin (1:200; Sigma), rabbit anti-nestin (1:200; Sigma), rabbit anti-Ki67 (1:200; Sigma) and rabbit anti-CD34 (1:200; Santa Cruz). After incubation with the primary antibodies, the cells were washed thoroughly and then incubated with appropriate secondary antibodies that were tagged with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Eugene, OR) for 1 h at RT. The cell nuclei were stained with 40 6-diamidino-2-phenylindole (DAPI, Sigma). To evaluate the percentage of Ki67 and nestin positive cells, 5–20 random fields per experiment were examined. The number of positive cells was expressed as a percentage of the total number of cells, which was evaluated by the DAPI stain.

4.2. Spinal cord surgery, cell transplantation and taxol treatment Female Sprague-Dawley rats (200–250 g) were used in accordance with local guidelines on the ethical use of animals and the National Institutes of Health Guidelines. After anesthesia by IP administration of 3.6% chloral hydrate (1 ml/100 g body weight), the animals received a dorsal laminectomy at the ninth thoracic vertebral level (T9) and the spinal cord with the dura mater was exposed. The dura matter covered spinal cord was crush-injured by spontaneously dropping a 10 g metal rod from a height of 7.5 cm using an NYU impactor. Immediately after injury, a second laminectomy was performed at thoracic level 12. A catheter was inserted through a small hole that was made in the dura mater and was directed to the injury site, ensuring that Taxol or PBS (control) was delivered at the lesion site. The SCI rats were randomly divided into 4 experimental groups: (I) the hUCMSCsþTaxol group (n¼ 8), which received Taxol via an Alzet osmotic mini pump (Alzet model 2004) and hUCMSCs transplantation into the spinal cord; (II) the Taxol group (n ¼8), which received Taxol via an Alzet osmotic mini pump and a PBS injection into the spinal cord; (III) the hUCMSCs group (n ¼8), which received PBS via an Alzet osmotic mini pump and hUCMSCs transplantation into the spinal cord; and (IV) the control group (n ¼8), which received PBS via an Alzet osmotic mini pump and a PBS injection into the spinal cord. For cell transplantation, a total of 2  105 cells, divided into two dosages, were transplanted into the injured spinal cord. The injections were made at 2 mm rostral and 2 mm caudal to the epicenter of the lesion at a depth of 1.2 mm. At each site, 2 ml of a cell suspension containing 105 cells or PBS was injected through a glass micropipette with a tip diameter of less than 60 mm at a rate of 0.4 ml/min. For Taxol infusion, Taxol was dissolved in a mixture of ethanol and cremophor EL oil (1:1; Sigma) and was administered through the intrathecal catheter connected to an osmotic mini-pump at 256 ng/d for 28 days (Hellal et al., 2011). All of the animals received daily intraperitoneal cyclosporine (Sandimmun; Novartis, Bern, Switzerland) at a dosage of 10 mg/kg from one day prior to transplantation until the animals were sacrificed.

4.3.

BBB open field locomotor test

Hindlimb function was assessed in an open field (100  60 cm plastic pool) using the BBB open field locomotor test. BBB

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tests were performed weekly, starting 1 day after transplantation and continuing for 4 weeks, by two examiners who were blinded to group identity.

4.4.

Tissue processing

The animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde, 1 week or 4 weeks (n¼ 4 for each group) after SCI for histological analysis. Dissected spinal cords were post-fixed overnight in 4% paraformaldehyde, incubated overnight in 30% sucrose, embedded in OCT compound (Sakura Finetechnical), and sectioned in the sagittal or axial plane at 12 mm on a cryostat (CM3050 S; Leica). The sections were stained with hematoxylin and eosin (HE) or were processed for immunohistochemistry followed by quantitative analysis. Tissue sections were stained with the following primary antibodies: rabbit anti–bIII-tubulin (1:1000, Abcam Inc., Cambridge, UK) for early neurons, rabbit antineurofilament 200 (NF200) (1:100, Sigma) for neurons and axons, rabbit anti-GFAP (1:1000, Millipore) for astrocytes, rabbit anti-CNPase (1:200, Sigma) for oligodendrocytes, rabbit anti-Ki67 (1:100, Millipore) to visualize proliferating cells, mouse anti-human nuclear protein (hNu, 1:100, Millipore) for the detection of transplanted human stem cells, rabbit anti-serotonin (5-HT) (1:1000, ImmunoStar, Hudson, WI) for the detection of raphespinal fibers, rabbit anti-Caspase-3 (1:100, sigma) for the detection of apoptotic cells and mouse anti-ED-1 (1:100, Millipore) for the detection of activated microglia/macrophages. The Fluoro-conjugated secondary antibodies we used were goat anti-mouse IgG-Alexa Fluor 488 or goat anti-rabbit IgG-Alexa 594 (Molecular Probes). After counterstaining with DAPI (Sigma), images were captured with a fluorescence microscope (model TCS SP2, Leica Instruments, Nusslosh, Germany).

4.5.

Cystic cavity assessment

Every tenth HE stained sagittal section (120 mm apart) of the central portions of the spinal cords was used to determine the cystic cavity volume in each group. At least 5 samples from each animal at 4 weeks post-injury were selected; thus, the central 600 mm portion of the lesion site was evaluated for the size of the cystic cavity. The area of cavitation of each section was measured using Image J software. Any necrotic tissue within the cavities was counted as a part of the lesion. The total spinal cord area of the sample was also measured. The volume was calculated using the Cavalieri method (Hains et al., 2004). V¼a  d, where (a) was the measured area and (d) was the intersection distance. A serial summation of the volumes was used to obtain the total volume (Vt ¼ V1þV2yþV10) of the cavitations (Vtc) and spinal cord tissue (Vtsp). Then, the total cavitation percentage (%Voltc) was determined according to the following equation: %Vtc ¼Vtc/Vtsp  100%.

4.6.

Quantitative analysis of cell survival

For quantitative analysis of the transplanted human stem cells in the spinal cord, 10 sections were collected from each

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rat spinal cord, 120 mm apart, and all of the hNuþ cells in each section were counted. Cell counts were adjusted using a previously described method (Hong et al., 2011). The number of cells was calculated using the method: N¼ n  10, where (n) was the number of hNuþ cells in a given section. A serial summation of N resulted in the total surviving cell number (Nt ¼N1þN2yþN10) in a given rat. Then, the percentage of cells that survived transplantation (%P) was determined according to the following equation: %P¼ Nt/200,000  100%.

4.7.

Quantitative analyses of stained tissue sections

To quantitatively compare the immunofluorescence staining intensities of GFAP immunoreactivity, we first selected a longitudinal spinal cord section with the largest cavity size as a reference and then included the two adjacent section (1 mm width) in the analysis (three sections per antibody staining). Three square regions of interest (ROIs, 400 mm  400 mm) were placed on each section along the rostral, lateral, and caudal borders of the spared spinal cord tissue. Special care was taken to use consistent ROI locations in different tissue sections and animals. Images of the ROIs were taken using the same detector settings for all sections and for all animals. The images were adjusted using the predetermined threshold setting in the Image J software. The average GFAP staining intensity was obtained. Quantification of the NF200þ fibers and ED-1þ microglia/macrophages was performed in three sections per animal, which were chosen by the same approach as described above. 5-HTþ fiber intensity was measured using Image J in the ventral gray matter of the lumbar intumescence (6–8 mm caudal from the epicenter of the lesion). The number of caspase-3þ apoptotic cells was manually counted in sagittal sections of the lesion epicenter at 400  magnification (n¼ 4 per group).

4.8.

Statistical analysis

Statistical analysis was performed using SPSS 13.0 for Windows. All of the data in this study are presented as means7SD. Data from more than 2 groups were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni post-hoc testing. The differences between groups were analyzed by Student’s t-tests. Differences were deemed statistically significant at Po0.05.

Acknowledgments We would like to acknowledge the staff at the Key Laboratory on Brain Function Repair and Regeneration of Guangdong for technical assistance.

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