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brain research 1608 (2015) 191–202

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miR-126 promotes angiogenesis and attenuates inflammation after contusion spinal cord injury in rats Jianzhong Hua, Lei Zenga, Jianghu Huanga, Guan Wanga, Hongbin Lub,n,1 a

Department of Spine Surgery, Xiangya Hospital, Central South University, Changsha 410008, PR China Department of Sports Medicine, Research Center of Sports Medicine, Xiangya Hospital, Central South University, Changsha 410008, PR China

b

art i cle i nfo

ab st rac t

Article history:

MicroRNAs are a class of small RNAs that regulate the expression of target mRNAs by

Accepted 13 February 2015

inhibiting translation or destabilizing target mRNAs. miR-126 is a microRNA that is highly

Available online 24 February 2015

enriched in endothelial cells. miR-126 has been found to promote angiogenesis and inhibit

Keywords:

vascular inflammation in endothelial cells by repressing three target genes Sprouty-related

microRNA

EVH1 domain-containing protein 1 (SPRED1), phosphoinositol-3 kinase regulatory subunit 2

Spinal cord injury

(PIK3R2), and vascular cell adhesion molecule 1 (VCAM1). Our previous study showed that

miR-126

the expression of miR-126 was downregulated after spinal cord injury (SCI). Therefore, we

Angiogenesis

wanted to examine whether upregulation of miR-126 could promote angiogenesis, inhibit

Inflammation

inflammation, and exert a positive effect on recovery after contusion SCI. In this study, we found that increased levels of miR-126 promoted angiogenesis, and inhibited leukocyte extravasation into the injured spinal cord, which was concurrent with downregulation of mRNA and protein expression of three validated miR-126 target genes, SPRED1, PIK3R2, and VCAM1. Moreover, a dose-dependent effect of miR-126 was observed in rescuing tissue damage and improving the functional deficit after SCI. Thus, the present study indicated that miR-126 played an important role in angiogenesis and inflammation after SCI. & 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.

Introduction

Contusive spinal cord injury (SCI) is a devastating neurological injury that causes progressive tissue loss, secondary to vascular dysfunction and inflammation at the injury epicenter (Bramlett and Dietrich, 2007; Fleming et al., 2006; Norenberg

et al., 2004). Endothelial cells and blood vessels at the epicenter of the injured spinal cord show degenerative changes minutes after a primary injury. Significant decreases in microvascular endothelial cells are observed subsequently because of necrotic or apoptotic cell death (Casella et al., 2006; Dohrmann et al., 1971). Hours later, leukocytes flood into the injured spinal cord

n

Corresponding author. Fax: þ86 731 84327332. E-mail address: [email protected] (H. Lu). 1 Postal address: Department of Sports Medicine, Research Center of Sports Medicine, Xiangya Hospital, Central South University, Changsha 410008, PR China. http://dx.doi.org/10.1016/j.brainres.2015.02.036 0006-8993/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 1 – Agomir-126 treatment reduced white matter loss following SCI. Compared to sham-operated (laminectomy only) rats with normal white matter (A), rats injected intravenously with saline over 7 d (B) had extensive loss of white matter as shown by myelin staining with Luxol fast blue in transverse sections at the injury epicenter. (C–E) Injection of agomir-126 at a concentration of 4, 20, or 100 nmol/mL increased the amount of spared white matter. (F–J) Adjacent sections of A–E were stained with hematoxylin-eosin (HE). Scale bar in (A) is 500 lm for (A–J). (K) The total white matter area at the injury epicenter (as a percentage of sham) showed that agomir-126 treatments improved white matter sparing compared to that with the saline at 7 d. Data are mean7SEM values. nPo0.05; nnPo0.01.

and produce oxidative and proteolytic enzymes such as myeloperoxidase (MPO) and NADPH, which provoke an inflammatory reaction (Bao et al., 2004; Brandes and Kreuzer, 2005; Taoka et al., 1997; Vaziri et al., 2004). Two to three days later, most blood vessels are destroyed and are lost in the lesion epicenter (Benton et al., 2008; Loy et al., 2002). While the surviving blood vessels become leaky (Whetstone et al., 2003), mediating leukocyte infiltration and activating macrophages that are derived from blood-borne monocytes and resident microglia (Donnelly and Popovich, 2008). An abundance of oxidative and proteolytic enzymes is released and causes further tissue damage. The injured spinal cord became swollen and edematous, exacerbating the ischemia (Casella et al., 2006; Loy et al., 2002), contributing to progressive degeneration and subsequent functional deficits (Hall and Springer, 2004; Norenberg et al., 2004; Tator and Fehlings, 1991; Tator and Koyanagi, 1997). Three to seven days after SCI, an angiogenic response is seen within the epicenter gray matter, which results in minimization of the size of the lesion and maintenance of viability;

however, this response might be too insignificant (Benton et al., 2008; Loy et al., 2002; Norenberg et al., 2004). In the past few years, studies that involved promotion of angiogenesis or attenuated the early inflammatory response demonstrated improved neurobehavioral recovery after SCI (Bethea, 2000; Gris et al., 2004; Han et al., 2010). However, previous attempts to develop therapies were confounded by complex mechanisms in clinical settings; nevertheless, a satisfactory therapy has been discussed here. MicroRNAs (miRs) constitute a class of small RNAs (approximately 20–25 nt) that regulate the expression of target mRNAs by inhibiting translation or destabilizing target mRNA. They greatly affect the regulation of signaling pathways that moderate common and specific functions across different cellular phenotypes and environments (Wu and Belasco, 2008; Zhao and Srivastava, 2007). miR-126 is a microRNA that is highly enriched in endothelial cells (Harris et al., 2008). Previous studies found that miR-126 plays an important role in vascular integrity and can promote angiogenesis during embryonic development and

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after injury (Fish et al., 2008; van Solingen et al., 2009; Wang et al., 2008). miR-126 was also found to inhibit vascular inflammation by targeting an intercellular adhesion molecule that mediated leukocyte infiltration (Harris et al., 2008). However, the specific roles of miR-126 in SCI have not yet been investigated. Our previous study found that the expression of miR-126 decreased after SCI (Hu et al., 2013). Therefore, in this study, we wanted to examine whether upregulation of the level of miR-126 could promote angiogenesis, inhibit inflammation, and exert a positive effect on recovery after contusion SCI. A previous study found that the angiogenic response within the epicenter gray matter diminished and disappeared concurrently with cystic cavity formation; therefore, therapies targeting angiogenic processes should be directed at the subacute interval after SCI (Loy et al., 2002). Therefore, we limited our intervention to 7 d to reduce the potential detrimental side effects of miR-126, which could occur at the same time.

2.

Results

2.1.

miR-126 reduced white matter loss following SCI

We determined effective doses of agomir-126 using white matter sparing at the epicenter as a measure because it correlates with locomotor function. Transverse sections in series of adjacent sections were used for measurements of white matter sparing and histological staining across all experiments. The injury epicenter was determined by the minimum area of myelin along the spinal cord axis. Compared to sham-operated (laminectomy only) rats with normal white matter (Fig. 1A), rats treated with saline for 7 d (Fig. 1B) showed extensive loss of the ventral white matter after injury. In agomir-126-treated groups, much of the ventral and lateral white matter was spared (Fig. 1C–E). The white matter area at the epicenter was greatly spared with agomir-126 at 4, 20, or 100 nmol/mL than with saline at 7 d postinjury (Fig. 1K). Intrathecal injections of miR-126 over 7 d at concentrations of 4, 20, or 100 nmol/mL showed that significantly better protection of white matter was obtained at 20 nmol/mL than at 4 nmol/mL and that the 100 nmol/mL dose did not further improve the outcome (Fig. 1K). Histological changes were visualized by HE staining of adjacent sections (Fig. 1F–J).

2.2.

miR-126 reduced locomotor deficits following SCI

miR-126 was tested for its effects on rescuing overground locomotion by using the BBB score (Fig. 2). After SCI, all rats were paralyzed in both hind limbs. Spontaneous recovery of function after SCI was observed among groups. Significant difference in functional testing among groups was not observed until 7 d; at that time, following SCI, rats administered intrathecal injections of agomir-126 at a concentration of 4, 20, or 100 nmol/mL showed more elevated locomotor recovery than the saline- or negative control-treated groups. Rats treated with agomir-126 at a concentration of 20 or 100 nmol/mL showed significantly better protection of white matter than those treated with the 4 nmol/mL dose, and there was no statistical significance between the 20 and 100 nmol/mL groups. These results correlated well with the results of epicenter white

193

Fig. 2 – Agomir-126 treatment provided lasting improvement in locomotor function following SCI in rats. After SCI, rats were intrathecally treated with saline, negative control (NC), and agomir-126 at a concentration of 4, 20, or 100 nmol/mL for 7 d. Hind limb recovery was assessed from 1 to 28 d after SCI by using BBB scoring. Bars represent means7SEM. n Po0.05, nnPo0.01 compared to saline.

matter sparing. A beneficial effect of miR-126 was also seen at 14 and 21 d and it was observed to last until 28 d. No statistically significant difference was observed between the saline-treated group and the negative control-treated group over 4 weeks of observation. Therefore, we used 20 nmol/mL agomir-126 for the subsequent experiments.

2.3.

miR-126 improved vascularity at the injury site

We used FITC-LEA to determine the presence of perfused blood vessels at the epicenter, as LEA only binds to the endothelial cells of perfused blood vessels. Twenty-four hours after SCI, compared to the results for the shamoperated rats (Fig. 3A), the number of LEA-labeled blood vessels around the epicenter greatly decreased in negative control and agomir-126-treated rats (Fig. 3B and E); furthermore, agomir-126-treated rats had more blood vessels than negative control-treated rats, although this difference was not statistically significant (P ¼0.08). Seven days after injury, the blood vessels in rats treated with the negative control did not increase (P40.05) (Fig. 3C). However, in agomir-126 treated rats, there was a significant increase in blood vessels at 7 d (Fig. 3G) compared to that at 24 h (Po0.05); more blood vessels were found in agomir-126 treated rats than negative control rats after 7-d treatment (Po0.01) (Fig. 3G).

2.4.

miR-126 reduced inflammation after SCI

Inflammation contributes to tissue damage after SCI. In this study, CD45 was used as a marker for extravasated leukocytes, and CD68 was used as a marker for activation of resident microglia and extravasated macrophages, which are major contributors to demyelination (Gris et al., 2004; Popovich et al., 1999; Shuman et al., 1997). In this study, three consecutive sections at 1-mm distances spanning 3 mm rostral to 3 mm caudal from the epicenter were selected for staining. Samples from the sham-operated rats

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Fig. 3 – Agomir-126 treatments improved vascularity after SCI. (A) LEA-labeled blood vessels in sham-operated rats had a normal appearance. (B) At 24 h following spinal cord injury, rats treated with the negative control (NC) had few blood vessels. (C) At 7 d after injury, rats treated with the negative control did not show an increase in blood vessels. (D) The inset box represents the region shown in A–C, E, and F. (E) Rats treated with agomir-126 for 24 h had more blood vessels, although this difference was not statistically significant (P ¼0.08). (F) Rats treated with agomir-126 for 7 d had a significant increase in blood vessels. Scale bar in (A) is 100 lm for (A–C, E, F). Data are mean7SEM. (G) The total area of blood vessels at the injury epicenter (as a percentage of sham) showed that agomir-126 treatments improved vascularity after injury. nPo0.05; nnPo0.01. dc: dorsal column, dh: dorsal horn.

showed little or no immunostaining for CD45 and CD68 (Fig. 4A and F). Twenty-four hours after SCI, there was no statistical significance between the negative control- and agomir-126treated groups (data not shown). Seven days after injury, samples from rats treated with the negative control showed abundant CD45-positive cells throughout the epicenters of the spinal cords (Fig. 4B and C) and in cells both rostral and caudal to it. This infiltration was markedly attenuated by agomir-126 (Fig. 4D and E). Similar results were observed with CD68 immunostaining (Fig. 4G–K). Peripheral leukocyte numbers were within the normal range in both the negative control and agomir-126 treated groups, at 7 d post-injury (Table 2).

2.5. The levels of three target genes were changed after miR-126 treatment SPRED1, PIK3R2, and VCAM1 are three validated target genes of miR-126. Previous studies found that miR-126 promotes angiogenesis by repressing SPRED1 and PIK3R2, and miR-126 could also inhibit vascular-related inflammation by repressing VCAM1 expression post-transcriptionally (Fish et al., 2008; Harris et al., 2008; Wang et al., 2008).

In our study, the level of miR-126 decreased at 1, 3, and 7 d after SCI (Fig. 5A). After 7 d treatment with agomir-126, the level of miR-126 in spinal cord increased (Fig. 6). To explore the possible mechanism underlying the effect of miR-126 on SCI, we examined mRNA levels of miR-126 target genes and corresponding protein levels using qRT-PCR and Western blotting after agomir-126 treatment. The mRNA levels of two target genes, SPRED1 and PIK3R2, at 1, 3, and 7 d after SCI were lower than those of sham-operated rats (Fig. 5B and C). Protein levels of these two targets correlated well with the mRNA changes (Fig. 7A). The mRNA and protein levels of these two genes sharply decreased at 1, 3, and 7 d after agomir-126 treatment compared to those with negative control-treated rats. The mRNA level of another target of miR-126, VCAM1, was elevated on 1, 3, and 7 d after SCI (Fig. 5D). The mRNA level of VCAM1 at 3 and 7 d was lower after treatment with miR-126 agomir than in the negative control-treated groups (Fig. 5D). However, we could not detect VCAM1 protein in either negative control or miR-126 agomir-treated groups. This lack of detection was not due to an ineffective antibody, since VCAM1 protein was detectible in positive control tissue (data not shown).

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Fig. 4 – Agomir-126 treatment reduced inflammation after SCI. (A) A transverse section through the entire spinal cord at the injury epicenter stained for CD45 showed no or little infiltration of leukocytes at 7 d in sham-operated rats. (B) Rats treated with the negative control (NC) for 7 d showed extensive infiltration of leukocytes post-injury. (D) Treatment with agomir-126 greatly reduced the infiltration. (C) and (E) are dual-stained, higher magnification images of (B) and (D) with 40 ,6-diamidino-2phenylindole dihydrochloride (DAPI). (G and I) CD68, a marker for activated microglia and macrophages, was similarly reduced by agomir-126. (F) Sham-operated rats exhibited no or little immunostaining. (H) and (J) are dual-stained highermagnification images of (G) and (I) with DAPI. Scale bar in (I) is 500 lm for (A, B, D, F, and G), and scale bar in (J) is 50 lm for (C, E, and H). (K) The total cross-sectional area of CD45-positive cells at the injury site showed that agomir-126 treatments reduced infiltration compared to that for the negative control at 7 d post-injury. The area was the sum of areas at 1 mm distance within a segment from 3 mm rostral to 3 mm caudal to the epicenter and was seen as a percentage of the negative control group within the experiment. The cross-sectional area of CD68-positive cells at the injury site showed that agomir-126 treatments reduced microglia/macrophage activation at all post-injury times. Data are mean7SEM values. nPo0.05; nnPo0.01 compared to the negative control.

We measured the phosphorylation status of ERK and AKT, two respective downstream targets of MAP and PI3 kinase pathways which can be activated by vascular endothelial growth factor stimulation. We found that the phosphorylation of ERK and AKT were lower than those of sham-operated rats at 7 d after SCI (Fig. 8). Compared with the results for the negative control, treatment with agomir-126 increased phosphorylation of ERK (p-ERK) and AKT (p-AKT) at 7 d (Fig. 8).

3.

Discussion

In accordance with the results of our previous study (Hu et al., 2013), we found that the expression of miR-126 was downregulated after SCI. Furthermore, in this study, we found that overexpression of miR-126 promoted angiogenesis, and inhibited leukocyte extravasation into the injured spinal cord, which was concurrent with downregulation of the mRNA and protein levels of three validated target genes of miR-126, SPRED1, PIK3R2, and

VCAM1. Moreover, a dose-dependent effect of miR-126 was observed while rescuing tissue damage and improving the functional deficit after SCI. Thus, the present study indicated that miR-126 plays an important role in angiogenesis and inflammation after SCI. miR-126 is a microRNA that is highly enriched in endothelial cells (Harris et al., 2008). miR-126, which is located in intron 7 of the EGF-like domain 7 (EGFl7) gene (Musiyenko et al., 2008), is regulated by the transcription factors Ets-1 and Ets-2 in endothelial cells (Harris et al., 2010). Previous studies found that miR-126 promoted stabilization and maturation of growing blood vessels by regulating angiopoietin-1 signaling (Sessa et al., 2012) and by repressing the p21-activated kinase 1 gene (Zou et al., 2011). miR-126 has been found to promote angiogenesis during embryonic development and after injury by targeting coding mRNAs of SPRED1 and PIK3R2, that is, inhibitors of angiogenic and cell survival signals in response to vascular endothelial cell growth factor (VEGF). Knockdown of miR-126 during zebrafish and mice embryogenesis has

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Fig. 5 – Agomir-126 increased mRNA expression of SPRED1, PIK3R2, and VCAM1. (A) The expression of miR-126 decreased at 3 d and 7 d post-injury compared to the sham. (B–D) The mRNA expression of SPRED1, PIK3R2, and VCAM1 after SCI was detected by qRT-PCR in samples collected from agomir-126 rats and negative control (NC) rats (n ¼4 per time point, respectively). Compared to the results for the negative control, treatment with agomir-126 significantly decreased SPRED1 and PIK3R2 expression at 1, 3, and 7 d. The expression of VCAM1 also decreased at 3 d and 7 d post-injury. Data are means7SEM values. nPo0.05, nnPo0.01.

Fig. 6 – Agomir-126 increased mRNA expression of miR-126. The expression of miR-126 decreased at 7 d post-injury compared to the sham. After 7 days treatment with agomir126 (20 nmol/mL), the level of miR-126 in spinal cord increased. Data are means7SEM values. nPo0.05, nnPo0.01.

been reported to result in delayed angiogenic sprouting, collapsed blood vessels, widespread hemorrhages, and partial embryonic lethality (Fish et al., 2008; Wang et al., 2008). Moreover, mice treated with a high dose of antagomir-126 have been found to exhibit a markedly reduced angiogenic response after ischemia of the left hind limb (van Solingen et al., 2009). In the current study, the number of blood vessels in the epicenters of injured spinal cords sharply decreased 24 h after SCI in rats, more blood vessels were observed at the

injury site at 24 h post-SCI for rats treated with agomir-126 than for the negative control group; however, this difference was not statistically significant (Student’s t-test, P¼ 0.08). At 7 d after SCI, no increase was observed in the number of blood vessels in the epicenters of injured spinal cords after 7d treatment with the negative control, which is similar to the natural recovery process. Rats that were treated with agomir126 for 7 d had a large increase in the number of blood vessels at the epicenter, numerous vessels reappeared in the dorsal column of injured spinal cord (Fig. 3F), where the vessels were lost 1 day after injury (Fig. 3E), suggesting that miR-126 could promote angiogenesis after SCI, as it does in vitro (Fish et al., 2008; Wang et al., 2008). Collectively, these data suggest that miR-126 promotes therapeutic angiogenesis following SCI. A previous study found that overexpression of miR-126 inhibited mRNA and protein expression of PI3KR2 and SPRED1 in endothelial cells (Fish et al., 2008). SPRED1 is a member of the sprouty and sprouty-related protein family, which are negative regulators of growth factor signaling (Wakioka et al., 2001). By binding and inactivating RAF1, an upstream kinase in the MAP kinase pathway, SPRED1 functioned as a membrane-associated suppressor of growth factor-induced ERK activation and blocked cell proliferation and migration in response to growth factor signaling, which are processes important for angiogenesis (Miyoshi et al., 2004; Nonami et al., 2004; Taniguchi et al., 2007). SPRED1 could also regulate the cytoskeleton organization (Johne et al., 2008; Miyoshi et al., 2004), a mechanism responsible for vasculature stabilization. PIK3R2 negatively regulated the activity of PI3

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Fig. 7 – Agomir-126 decreased protein levels of SPRED1 and PIK3R2. After SCI, the protein levels of SPRED1 and PIK3R2 were detected by Western blot (A) in samples collected from agomir-126 rats and negative control (NC) rats (n¼ 4 per time point, respectively), and the intensity of each band was estimated by densitometric analysis (B). Compared with the results for the negative control, treatment with agomir-126 decreased SPRED1 levels at 1, 3, and 7 d. The PIK3R2 level at 3 d and 7 d postinjury also decreased after agomir-126 treatment, compared to the results for the negative control. Data are means7SEM values. nPo0.05, nnPo0.01.

Fig. 8 – Agomir-126 increased protein levels of pERK and pAKT. After SCI, the protein levels of pERK/ERK and pAKT/AKT were detected by Western blot in samples collected from agomir-126 rats and negative control (NC) rats (n ¼ 4 per time point, respectively). Compared with the results for the negative control, treatment with agomir-126 (20 nmol/mL) increased phosphorylation of ERK (p-ERK) and AKT (p-AKT) at 7 d. Data are means7SEM values. nPo0.05, nnPo0.01.

kinase, a kinase important in the Akt pathway, which is related to an antiapoptotic effect (Ueki et al., 2003). ERK and AKT are two respective downstream targets of MAP and PI3 kinase pathways which can be activated by vascular endothelial growth factor stimulation. In the current study, we found that the phosphorylation of ERK and AKT were decreased after SCI. Treatment with agomir-126 increased phosphorylation

of ERK and AKT. We also found that the expression of SPRED1 and PIK3R2 were sharply decreased after agomir-126 treatment. Thus, these findings suggest that miR-126 could moderate the expression of these genes after SCI. Further studies should be done to get deep insight into the mechanisms of miR-126 and its target genes after SCI. We also observed an interesting phenomenon in which expression level of the miR-126 and its

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target genes SPRED1 and PIK3R2 all decreased after SCI, suggesting that the modulation of SPRED1 and PIK3R2 in SCI may involve multiple mechanisms in addition to miR-126. Therefore, more work should be performed to gain insights into the exact mechanism controlling SPRED1 and PIK3R2 expression. Therapies enhancing angiogenesis may have differing outcomes. Some researchers found that treatment with VEGF, a factor enhancing angiogenesis, improved the functional outcome and decreased secondary degeneration after SCI (Liu et al., 2010; Widenfalk et al., 2003). Other researchers found that therapies promoting angiogenesis by activating Notch signaling had no effect on hind limb locomotor recovery after SCI (Fassbender et al., 2011). Some studies have even reported that therapies enhancing angiogenesis have detrimental effects that exacerbate tissue damage and vascular permeability (Benton and Whittemore, 2003; Patel et al., 2009). In this study, the current data contribute evidence to the idea that combining angiogenesis with vascular stabilization may have potential therapeutic applications following SCI, which correlates with other studies (Han et al., 2010; Herrera et al., 2010). In addition to promoting angiogenesis, miR-126 also has anti-inflammatory activity. VCAM1 is an adhesion molecule expressed by activated endothelial cells, which mediate leukocyte adherence to endothelial cells (Alon et al., 1995; Osborn et al., 1989). By suppressing VCAM1 expression, miR126 activity decreases leukocyte interactions with endothelial cells and attenuates vascular inflammation after injury (Asgeirsdottir et al., 2012; Harris et al., 2008). In the current study, rats treated with the negative control had abundant leukocytes and activated microglia/macrophages at the epicenter of the injured spinal cord on the 7th day. Reduced leukocyte infiltration and inflammation responses were seen after treatment with agomir-126 for 7 d, which suggests that miR-126 could inhibit leukocyte infiltration and reduce inflammation after SCI. The expression of VCAM1 decreased after agomir-126 treatment, which suggests that miR-126 can moderate VCAM1 expression in an SCI model. Infiltrating leukocytes and inflammatory cells may act counter-productively after SCI. Apart from the detrimental effects of some leukocytes, such as the early arriving neutrophils and activated macrophages (Donnelly and Popovich, 2008; Weaver et al., 2005), infiltrating leukocytes such as the M2-type macrophages showed beneficial effects that promoted cell survival and regeneration after SCI (Kigerl et al., 2009). Thus, further work should be performed to better understand the effects of miR-126 on sub-classes of these cells. Depending on the different cell types and the local environment, expression and signaling of miR-126 may vary greatly. Apart from these findings described above, miR-126 was found in neurons (Kim et al., 2014a, 2014b) and could target other pathways such as IGF-1 signaling, which may promote neurite sprouting, synapse formation, and neuronal survival in the central nervous system (Ryu et al., 2011; Zhang et al., 2008). Thus, more research should be focused on this area. In conclusion, in this study, we found that miR-126 expression decreased after SCI, but that increasing the levels of miR-126 rescued more white matter, promoted angiogenesis, reduced inflammation, and improved function deficit, which was concurrent with downregulation of expression of SPRED1, PIK3R2, and VCAM1 target genes; however, more

research needs to be performed to understand the exact mechanisms underlying the effect of miR-126 in SCI.

4.

Experimental procedures

4.1.

Animals and overall design

A total of 152 Sprague Dawley (SD) male rats were used (weight, 180–220 g; Central South University, Hunan Province, China) and were age- and weight-matched between groups Table 1 – Biological replicates (number of rats) in each of the experiments. Experiment

Control

n

Treatment

n

7-Day injury (LFB,HE)

Sham Saline Sham Saline

4 4 6 6

Agomir:3 dose

12

Agomir:3 dose Negative control Agomir

18 6

Negative control Agomir

4

Negative control Agomir Negative control Agomir Negative control Agomir

4

Negative control Agomir

8

Negative control Agomir

8

Negative control

8

28-Day injury (BBB)

1-Day injury (FITCLEA)

7-Day injury (FITCLEA)

Sham

4

1-Day injury (IF)

7-Day injury (IF)

Sham

4

1-Day injury (PCR, WB)

3-Day injury (PCR, WB)

7-Day injury (PCR, WB)

Sham

8

4

4

4 4 4 4 8

8

8

LFB: Luxol fast blue staining for detecting myelin in white matter tracts at 7 d post-injury. HE: Hematoxylin-eosin staining to detect pathological changes at 7 d after injury. BBB: Basso, Beattie, and Bresnahan score for measuring hind limb locomotor function; BBB scores were measured at 1, 3, 7, 14, 21, and 28 d post-injury. FITC-LEA: Fluorescein isothiocyanate-conjugated Lycopersicon esculentum agglutinin lectin for detecting blood vessel density at 1 and 7 d post-injury. IF: Immunofluorescence staining for analyzing the marker of leukocytes (CD45) and the marker of activated microglial/macrophages (CD68) at 1 and 7 d post-injury. PCR: Real-time fluorescence quantitative RT-PCR for determining the levels of miR-126 and mRNA of its target genes SPRED1, PIK3R2, and VCAM1 at 1, 3, and 7 d post-injury. WB: Western blot analysis for quantifying the protein levels of SPRED1, PIK3R2, VCAM1, AKT, pAKT, ERK and pERK post-injury.

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Table 2 – Agomir-126 and negative control did not affect leukocyte counts.

Leukocytes total (109/L) Neutrophils (109/L) Lymphocytes (109/L) Monocytes (109/L) Eosinophils (109/L) Basophils (109/L) Erythrocytes (1012/L) Thrombocytes (109/L)

Normal range

Saline

Negative control

Agomir-126

1.8–11.8 0.1–3.2 0.9–9.8 0.0–0.5 0.0–0.2 0.0–0.2 6.04–11.1 276–1102

5.2570.82 1.5770.24 4.3370.69 0.270.03 0.170.03 0.170.02 7.3271.23 6277173

5.6270.69 1.6670.33 4.8271.03 0.370.02 0.170.02 0.170.02 7.8370.83 7287105

6.1771.08 1.5170.28 4.2770.83 0.270.03 0.170.02 0.170.02 8.1771.17 6537132

Rats were treated with saline, negative control or agomir-126 (n ¼8) for 7 days after spinal cord injury and 1 mL blood was drawn from the heart for blood cell analysis before perfusion-fixation. Low red blood cell counts are probably due to bleeding caused by surgery. Data are mean7SEM values.

within an experiment. All animal procedures were approved by the Laboratory Animal Users Committee of Central South University. An overview of all experimental groups is presented in Table 1. We first determined whether upregulating miR-126 with agomir-126 could decrease the extent of injury and optimal dose for protecting white matter at 7 d post-injury. A dose-response study for white matter rescue was performed at the same time. Next, we determined whether a 7-d treatment with agomir-126 would improve locomotor function over 4 weeks. Additional groups of rats were used to investigate the effects and mechanisms of miR-126 on vascular and inflammatory responses at 1, 3, and 7 d. All surgeries, behavioral measurements, and quantification of histological results were performed by testers blinded to the treatments. Treatments were assigned in a randomized order and were prepared by someone other than the surgeon.

4.2.

Surgery

Rats were anesthetized by intraperitoneal injection of 0.3 mg/g body weight chloral hydrate (Kermel, Tianjing, China), and their backs were shaved and cleaned. After a midline incision and laminectomy at vertebral level T9/T10, spinal cord contusions were induced using a modified Allen’s weight drop apparatus (8 g weight at a vertical height of 40 mm, 8 g  40 mm). A partial laminectomy at T12/T13 was performed for the placement of an intrathecal catheter. miR-126 agomir and miR-126 agomir negative control (NC) were from RiboBio (Guangzhou, China). In brief, the agomir-126 (50 -UCG UAC CGU GAG UAA UAA UGC G30 ) or agomir-126 negative control (50 -UCA CAA CCU CCU AGA AAG AGU AGA-30 ), which was dissolved in saline (0.9%), was continuously delivered (1 μL/h) into the intrathecal space using subcutaneously implanted osmotic mini-pumps (Alzet 1030D, CA, USA) connected to a subdural-implanted catheter, as previously described (Liebscher et al., 2005). Each pump was primed overnight at 37 1C to ensure immediate delivery after implantation. After the implantation, muscles were sutured in layers, the skin incision was closed with 3-0 silk threads, and 5 mL of lactated Ringer’s solution was administered intraperitoneally. All surgeries were performed in a warm environment to maintain body temperature. After the surgery, rats were placed on fresh dry cages and given free access to food and water. Penicillin G (40,000 u, i. m.) was administered daily for

3 d to prevent infection. Bladders were manually expressed twice daily until full voluntary or autonomic voiding was obtained. To determine the effects of miR-126 on the number of peripheral leukocytes, 1 mL of blood was drawn from the heart just before perfusion-fixation in rats that had been treated with negative control or agomir-126 for 7 d. Blood counts were performed with an automated hematology analyzer (Beckman Coulter, USA).

4.3.

Functional testing

Functional recovery after SCI was determined at 1, 3, 7, 14, 21, and 28 d post-injury by two independent and well-trained testers blinded to treatment, using open-field overground locomotor performance according to Basso, Beattie, and Bresnahan (BBB) scores (Basso et al., 1995). The final score for each animal was obtained by averaging the values from both testers. Rats with perineal infections, wounds in the limbs, or tail and foot autophagia were excluded from the test.

4.4.

Histological procedures

Rats were perfused transcardially with phosphate-buffered saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Subsequently, the spinal cords were carefully dissected out, and 10-mm segments containing the injury site were post-fixed for 24 h at 4 1C. To detect myelin in white matter tracts, 1 of every 5 of the transverse sections at each rostro-caudal 1-mm level was stained with Luxol fast blue, as previously described (Han et al., 2010). After fixation, spinal cords were embedded in paraffin, sectioned at 5-mm thickness, and then mounted onto charged microscope slides for further use. Slides were placed in xylene at room temperature for two 20-min periods, passed through graded ethanol solutions (twice each in 100% and 95% ethanol, once in 70% ethanol, and twice in double-distilled H2O), and stained with 0.1% Luxol fast blue in 0.5% glacial acetic acid and 95% ethyl alcohol overnight at 56 1C. Next, the slides were gently washed in 95% ethanol to rinse off excess stain and then briefly rinsed in doubledistilled water. The slides were differentiated in 0.05% lithium carbonate solution, differentiated in 70% ethanol, briefly rinsed in double-distilled water, dehydrated briefly through graded ethanol solutions, cleared through xylene,

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and coverslipped. The injury epicenter was determined for each rat on the basis of the rostro-caudal level that contained the least amount of spared myelin per transverse section, to align all the histological measurements for each rat. Adjacent sections were stained with hematoxylin-eosin (HE) staining. To label the perfused blood vessels, the jugular vein was exposed and injected with 500 mg/250 mL fluorescein isothiocyanate-conjugated Lycopersicon esculentum agglutinin lectin (LEA; Sigma, USA) at 30 min before euthanasia. After intravenous injection, LEA binds only to endothelial cells of perfused blood vessels (Mazzetti et al., 2004), allowing for simultaneous detection of endothelial cell survival in perfused blood vessels. Ten consecutive 30-μm transverse sections per millimeter along the spinal cord axis were cut and mounted onto charged microscope slides for further detection. To detect immunofluorescent staining of CD45 (leukocytes) and CD68 (activated microglia/macrophages), spinal cords were placed in 30% phosphate-buffered sucrose overnight at 4 1C after fixation. Twenty consecutive 10-mm transverse sections at each 1-mm rostro-caudal level along the spinal cord axis were cut on a cryostat and thaw-mounted onto charged microscope slides. The sections were stored in sequence at –20 1C for further use. Slides were warmed for 30 min on a slide warmer, a ring of wax was applied around the sections with a PAP pen (Zsbio, Beijing, China), and the slides were rinsed in 0.1 M Trisbuffered saline (TBS) for 15 min. After blocking non-specific staining with 10% donkey serum in TBS containing 0.3% Triton X-100 for 1 h at room temperature, sections were incubated overnight at 4 1C in TBS, containing 5% donkey serum, mouse anti-CD45 (1:100; catalog number 05-1410, Millipore, Temecula, CA, USA), and mouse anti-CD68 (1:100; catalog number MCA341GA, ABD Serotec, Kindlington, Oxford, UK). Purified rat immunoglobulin G (IgG) was used as a control at the same concentrations instead of the primary antibody. Next, the sections were incubated in TBS-Triton containing 5% donkey serum with secondary antibodies (1:400; Jackson, USA) for 1 h at room temperature. Subsequently, sections were stained with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, D9542, USA) for 10 min at room temperature. Finally, the sections were cover-slipped with antifade Gel/Mount aqueous mounting media (Beyotime, Shanghai, China). Between steps, sections were washed 3 times for 10 min in TBS. For normalizing all histological measurements, four sham-operated rats received a laminectomy only and were analyzed 7 d later.

4.5.

Real-time polymerase chain reaction

From 10-mm long spinal cord segments containing the injury epicenter, total RNA was extracted with TRIzol (Invitrogen, CA, USA) according to manufacturer’s instructions. To analyze microRNA expression by quantitative real-time PCR (qRT-PCR), RNA was reverse-transcribed using microRNA-specific primers from RiboBio. Real-time PCR was performed on diluted samples with miR-U6 as an internal control. For quantification of mRNA expression, first-strand synthesis was performed using a PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan). Primers were designed and supplied by Sangon Biotech (Shanghai, China). Gene expression changes were quantified using the delta-delta CT method. The expression of GAPDH was used as an internal

control. Reverse transcription (RT) reactions was performed on the Applied Biosystems 9700 Thermocycler using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) at 37 1C/42 1C for 15 min and 85 1C for 5 s. Real-time PCR reactions were conducted on the iCycler iQ Real-time detection System (Bio-Rad) at 95 1C for 30 s, followed by 40 cycles each of 95 1C for 5 s and 60 1C for 30 s, using SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan).

4.6.

Western blot

Rats were euthanized with an overdose of chloral hydrate. After the animals (n¼4 per group) were sacrificed, 10-mm long segments of spinal cord encompassing the injury site were then harvested for Western blot analyses. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Each protein sample (30 μg) was loaded for the assay. Nonspecific bands were blocked in TBS-T (25 mM Tris (Sigma), 150 mM NaCl, 0.05% Tween 20, pH 7.5) containing 5% non-fat milk for 1 h at room temperature. Membranes were subsequently incubated with the primary antibodies of rabbit anti-Sprouty-related EVH1 domaincontaining protein 1 (SPRED-1) polyclonal antibody (1:200; ab77079, Abcam, USA), rabbit anti-phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) polyclonal antibody (1:200; ab131067, Abcam), rat anti-vascular cell adhesion molecule 1 (VCAM1) monoclonal antibody (1:50; ab78712, Abcam), rabbit anti-ERK1/2 polyclonal antibody (1:100; Abcam), rabbit antiphospho-ERK1/2 (Thr202/Tyr204) antibody (1:200; Cell Signaling), rabbit anti-AKT1/2/3 polyclonal antibody (1:100; Abcam), rabbit anti-phospho-AKT1/2 antibody (1:200; Cell Signaling) at 4 1C and followed by anti-mouse IgG horseradish peroxidase conjugate secondary antibody (1:2000; Jackson) for 1 h at room temperature. The immunocomplexes were visualized by chemiluminescence using an ECL kit (Beyotime, Shanghai, China). The film signals were digitally scanned and then quantified using the Quantity One 4.6.2 software (Bio-Rad, USA). An antibody for GAPDH (1:35,000; Sigma, USA) was used as an internal control. Data have been provided in terms of the mean7SD of the percentage ratio of the control.

4.7.

Statistics

All data were analyzed using the IBM SPSS 19.0 statistical software. Two-way repeated measures ANOVA with the posthoc Tukey test was used to analyze the differences in BBB scores among groups over time. Statistically significant differences between two groups were determined by a two-tailed ttest. One-way ANOVA followed by post-hoc Tukey analysis was performed to compare groups of three or more. In cases where data were not normally distributed, the Kruskal–Wallis test was used. A value of Pr0.05 was considered statistically significant. Values are presented as mean7SEM.

Acknowledgments This work was supported by the National Natural Science Foundation of China (nos. 81171698 and 81371956).

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