• Users Online: 2407
  • Home
  • Print this page
  • Email this page

 Table of Contents  
RESEARCH ARTICLE
Year : 2018  |  Volume : 13  |  Issue : 8  |  Page : 1447-1454

Local inhibition of matrix metalloproteinases reduced M2 macrophage activity and impeded recovery in spinal cord transected rats after treatment with fibroblast growth factor-1 and nerve grafts


1 Department and Institute of Pharmacology, National Yang-Ming University; Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan, China
2 Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan, China
3 Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan, China
4 Department and Institute of Pharmacology, National Yang-Ming University; Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital; Center for Neural Regeneration, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan, China

Date of Acceptance26-May-2018
Date of Web Publication10-Aug-2018

Correspondence Address:
Henrich Cheng
Department and Institute of Pharmacology, National Yang-Ming University; Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital; Center for Neural Regeneration, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan
China
Meng-Jen Lee
Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan
China
Login to access the Email id

Source of Support: This work was supported by the National Science Council (102-2320-B-324-001), China. This study was also supported by grants from Taipei Veterans General Hospital (V103E6-001 & V104E6-001) and by grants (MOST 104-2314-B-010-012-MY3, MOST 105-2314-B-010-013-MY2 and MOST 106-2632-B-324-001) from the Ministry of Science and Technology in Taiwan, China, Conflict of Interest: None


DOI: 10.4103/1673-5374.235302

Rights and Permissions
  Abstract 


Alternatively activated macrophages (M2 macrophages) promote central nervous system regeneration. Our previous study demonstrated that treatment with peripheral nerve grafts and fibroblast growth factor-1 recruited more M2 macrophages and improved partial functional recovery in spinal cord transected rats. The migration of macrophages is matrix metalloproteinase (MMP) dependent. We used a general inhibitor of MMPs to influence macrophage migration, and we examined the migration of macrophage populations and changes in spinal function. Rat spinal cords were completely transected at T8, and 5 mm of spinal cord was removed (group T). In group R, spinal cord-transected rats received treatment with fibroblast growth factor-1 and peripheral nerve grafts. In group RG, rats received the same treatment as group R with the addition of 200 μM GM6001 (an MMP inhibitor) to the fibrin mix. We found that MMP-9, but not MMP-2, was upregulated in the graft area of rats in group R. Local application of the MMP inhibitor resulted in a reduction in the ratio of arginase-1 (M2 macrophage subset)/inducible nitric oxide synthase-postive cells. When the MMP inhibitor was applied at 8 weeks postoperation, the partial functional recovery observed in group R was lost. This effect was accompanied by a decrease in brain-derived neurotrophic factor levels in the nerve graft. These results suggested that the arginase-1 positive population in spinal cord transected rats is a migratory cell population rather than the phenotypic conversion of early iNOS+ cells and that the migration of the arginase-1+ population could be regulated locally. Simultaneous application of MMP inhibitors or promotion of MMP activity for spinal cord injury needs to be considered if the coadministered treatment involves M2 recruitment.

Keywords: spinal cord injury; fibroblast growth factor-1; matrix metalloproteinase; GM6001; macrophage


How to cite this article:
Chiu CW, Huang WH, Kuo HS, Tsai MJ, Chen CJ, Lee MJ, Cheng H. Local inhibition of matrix metalloproteinases reduced M2 macrophage activity and impeded recovery in spinal cord transected rats after treatment with fibroblast growth factor-1 and nerve grafts. Neural Regen Res 2018;13:1447-54

How to cite this URL:
Chiu CW, Huang WH, Kuo HS, Tsai MJ, Chen CJ, Lee MJ, Cheng H. Local inhibition of matrix metalloproteinases reduced M2 macrophage activity and impeded recovery in spinal cord transected rats after treatment with fibroblast growth factor-1 and nerve grafts. Neural Regen Res [serial online] 2018 [cited 2022 Jan 26];13:1447-54. Available from: http://www.nrronline.org/text.asp?2018/13/8/1447/235302




  Introduction Top


Nerve regeneration in the central nervous system (CNS) is difficult due to the excessive amount of inhibitory extracellular molecules and the weak neurite outgrowth of severed axons; however, recent progress has been made by enzymatic digestion of inhibitory molecules (Bartus et al., 2014), supplementation with stem cells, and promotion of neurite outgrowth (Ghannam et al., 2010). Macrophages are heterogeneous cell populations with distinct functions in tissue repair or injury. The polarization of macrophages occurs in response to the microenvironment via the expression of distinct functional programs. M1 (classically activated) macrophages can be induced by interferon-γ, lipopolysaccharide, tumor necrosis factor and granulocyte-macrophage colony-stimulating factor. M1 macrophages mediate resistance against bacterial infection, parasites, and tumor (Gordon, 2003; Mantovani et al., 2004). M2 (alternatively activated) macrophages can be induced by interleukin (IL)-4, IL-13, IL-10 and glucocorticoids (Franco and Fernandez-Suarez, 2015). Although M2 macrophages can be divided into subsets, they generally express high levels of mannose receptors, galactose receptors, and arginase-1 (Arg1) (Cherry et al., 2014). Arg1 converts arginine into ornithine and polyamines (Mantovani et al., 2013). Postinjury CNS repair is a process that involves the balanced action of M1/M2 macrophage populations (David and Kroner, 2011; Shechter and Schwartz, 2013). Following SCI, M1 macrophages produce high levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines (such as IL-1β, tumor necrosis factor alpha and IL-6), which frequently cause tissue damage and axonal retrasction (Busch et al., 2009). In contrast, M2 macrophages secrete growth factors, such as glial cell-derived neurotrophic factor, brain-derived neurotrophic factor (BDNF), and oncomodulin, and are involved in tissue repair (Mantovani et al., 2013; Gensel and Zhang, 2015; Hu et al., 2015).

One treatment that uses peripheral nerve autografts to bridge a complete spinal cord lesion in combination with fibroblast growth factor (FGF)-1 leads to functional locomotor recovery in a complete spinal-transection rodent model (Cheng et al., 1996). The Basso, Beattie and Bresnahan (BBB) score improved, and the detection of somatosensory-evoked potentials and motor-evoked potentials indicated that both sensory and motor information can cross the damaged site (Lee et al., 2002). The regeneration of axons through the graft and into the distal stump was demonstrated by retrograde labeling in the cortex and brain stem nuclei and by anterograde labeling in the distal stump (Cheng et al., 1996; Lee et al., 2004). Increased numbers of M2 macrophages were evident (Kuo et al., 2011), and the neurite inhibitory molecule chondroitin sulfate proteoglycan and glial reactive scar were also reduced in the treated rats (Lee et al., 2011). During the normal course of SCI, M1 macrophages sustain activities beyond the acute stage. M2 macrophages are present during the initial stage but are outnumbered by M1 macrophages at the injury site after the acute stage (Kigerl et al., 2009). Promoting an alternative macrophage phenotype may be a useful strategy for repairing a spinal cord injury. However, a method of consistently inducing a sustained, dominant M2 phenotype is presently unknown. Functional recovery in the aforementioned combination-treated rats suggests that the study of the origin and migration pathway of the M2 population may provide insight into how to harness the beneficial effects of M2 macrophages.

Following injury, the damaged tissue releases chemokines and attracts monocytes/microglia, which migrate to the lesioned area and become activated macrophages. As the migration of myeloid cells or inflammatory macrophages is matrix metalloproteinase (MMP) dependent (Gong et al., 2008; Letellier et al., 2010), in this study, we used a general MMP inhibitor to block the migration of macrophages to study the activities of macrophages for functional recovery observed in spinal cord-transected rats treated with fibroblast growth factor-1 and peripheral nerve grafts. MMP2 and MMP9 were reported to be regulated by factors that promote conversion of the M1 or M2 phenotype (Lolmede et al., 2009; Veeravalli et al., 2009; Huang et al., 2012). The levels of these two MMPs were examined in different treatment groups to determine whether there is an association with the M1 or M2 phenotype or with functional recovery.


  Materials and Methods Top


Experimental animals

Young adult female Sprague-Dawley (SD) rats (250–280 g, ~8 weeks old) were used in this study. Surgical procedures, postoperative care, and monitoring were conducted as previously described (Kuo et al., 2007, 2011). The animal use protocol listed below was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Veterans General Hospital, China (IACUC approval number VGHTPE IACUC 2015-167). The rats were randomly separated to groups: N, T, R, RG, and U (see following). Group N is for rats those spinal cords were not transected. For group T spinal cords were completely transected at T8, and 5 mm of spinal cord tissue was removed. In group R, spinal cord transected rats received a combined treatment with FGF-1 and a peripheral nerve graft that was developed by Cheng (Cheng et al., 1996). Briefly, autologous peripheral intercostal nerve segments combined with fibrin glue mixture (100 μL Hanks' balanced salt solution mixed with 10 μg fibrin glue and 1 μg FGF-1) (Beriplast P; Behringwerke Medical Inst., Marburg, Germany) was implanted to bridge a 5-mm gap in the transected spinal cord. In addition, to study the effects of MMP-2 and MMP-9 inhibition, GM6001 (an MMP inhibitor; MilliporeSigma) was mixed in the fibrin glue to a concentration of 200 μM in addition to FGF-1 (group RG). For quantitative real-time polymerase chain reaction (PCR) analysis, transection supplemented with glue (group U) was used as a control group for normalization. Manual emptying of the bladder was performed twice daily.

RNA isolation, reverse transcription, and quantitative real-time PCR analysis

On specified days following injury, rats were sacrificed with an overdose of anesthetic sodium pentobarbital intravenously (Health-Tech Pharmaceutical Co., Ltd., Taiwan, China). Then, 5 mm (length) of spinal cord surrounding the lesioned area was collected for RNA isolation, as shown in [Figure 1]. Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. First-strand cDNA synthesis was primed with oligo dT and then reverse-transcribed using Superscript Reverse Transcriptase (Invitrogen) as previously described (Kuo et al., 2011). cDNA levels were quantified using MMP-2, MMP-9, Arg1, iNOS, and BDNF primer pairs [Table 1] with a QuantiTect SYBR Green PCR Kit (Qiagen) on a Light Cycler 480 (Roche Applied Science). Ribosomal protein L13A (RPL13A) was used as the reference gene in each set of reactions (Jesnowski et al., 2002; Tian et al., 2007). Melting curve analyses verified the formation of the single desired PCR product. Each sample was run in duplicate. The relative differences in expression between the groups were analyzed on the basis of cycle time values using the comparative threshold cycle (Ct) method (Livak and Schmittgen, 2001), in which the Ct value indicates the cycle in which there is the first demonstrable increase in SYBR Green fluorescence above the threshold. The target gene quantity was normalized to the reference gene using the formula: 2–(Ct(target)–Ct(reference)).
Figure 1: Matrix metalloproteinase (MMP)-9 mRNA expression was upregulated in the graft area of the repaired spinal cord in rats.

Click here to view
Table 1: Sequences of quantitative polymerase chain reaction primer sets

Click here to view


Immunofluorescence staining

At 10 days postinjury, rats received an overdose of sodium pentobarbital and were intravascularly perfused with 0.9% saline and 4% paraformaldehyde in phosphate buffered saline (PBS). The spinal cords were removed from the experimental animals, postfixed in 4% paraformaldehyde overnight, rinsed, cryoprotected in graded sucrose, embedded in an Tissue-Tek optimum cutting temperature compound (Sakura Fintek USA) at –20°C and longitudinally sectioned (20 μm thick) for staining. The primary antibodies included mouse anti-ED1 (1:250, Biorad, Hercules, CA, USA) and goat anti-Arg1 (1:100, Santa Cruz Biotechnology Inc, Dallas, TX, USA) at 4°C overnight, and nuclear staining was achieved via incubation with 1 μg/mL DAPI for 1 minute. The secondary antibodies were donkey anti-mouse, and donkey anti-goat (1:250, Invitrogen, Carlsbad, CA, USA), incubated at 4oC for 1 hour. Photo images were obtained from stained slides of sections using an Axioscope fluorescence microscope and a CCD camera (Carl Zeiss AG, Oberkochen, Germany). The images were arranged in Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA, USA). The immunoreactivities of Arg1 and ED1 were quantified in 3 sections from each rat using NIH ImageJ 1.44d software (software developed by National Institutes of Health, USA); three to four images were obtained through random sampling inside the graft area of each section. The threshold values of immunoreactivities were constant in all analyzed images.

Western blotting analysis

On days 10 and 14 post-injury, spinal cord segments (one-centimeter-long segments) were homogenized in lysis buffer containing 200 mM Tris-HCl (pH 6.8), 10% SDS, 5 mM ethylene glycol tetraacetic acid (EGTA), 5 mM EDTA, 10% glycerol, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were centrifuged at 13,000 × g for 30 minutes. The supernatant fractions were collected for Western blotting analysis as previously described (Kuo et al., 2007). Proteins (20 μg/lane) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked in PBS that contained 3% skim milk for 30 min. Each blot was incubated overnight at 4°C with a primary antibody against goat anti-β-actin (1:1000, Sigma), and goat anti-Arg1 (1:500, Santa Cruz Biotechnology Inc.). After washing in PBS, the membranes were incubated with donkey anti-goat IgG HRP (horseradish peroxidase)-conjugated secondary antibody (1:5000, Santa Cruz Biotechnology Inc) or goat anti-mouse IgG HRP-conjugated secondary antibody (1:5000, Santa Cruz Biotechnology Inc) for 2 hours at room temperature. Subsequent visualization was performed using SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL, USA).

In situ zymography

Rats received an overdose of sodium pentobarbital and were intravascularly perfused with 0.9% saline. The spinal cord samples were removed from the experimental animals, immediately embedded into OCT compound at –20°C and sectioned longitudinally on a cryostat (Leica Biosystems, Buffalo Grove, IL, USA). The sections were incubated in an in situ zymography reaction buffer consisting of 0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, and 0.1 mM NaN3 and 40 μg fluorescein-conjugated DQ gelatin (Molecular Probes, Eugene, Oregon, USA) at 37°C for 1 hour in the dark. The gelatin with a fluorescent tag remained caged until cleavage by MMP-2 or MMP-9. The reaction product was visualized using fluorescence microscopy (Carl Zeiss AG, Oberkochen, Germany).

Gelatin zymography

Rats received an overdose of sodium pentobarbital and were intravascularly perfused with 0.9% saline. One-centimeter spinal cord segments including grafts and caudal stumps of spinal cord were weighed and homogenized by sonication (1:10, w:v) in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Soluble and insoluble extracts were separated by centrifugation, and the supernatant was stored at –20°C. Equal amounts of supernatant were separated using a 10% SDS gel with 0.1% gelatin (Invitrogen). After separation, the gel was renatured by washing in 2.5% Triton X-100 and incubating in substrate buffer (50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, and 0.01% sodium azide) for 48 hours at 37°C to enable MMP-2 and MMP-9 to cleave the gelatin. Each gel was then stained with Coomassie blue for 1 hour and destained in a solution of 30% methanol and 10% acetic acid. Negative staining indicated the locations of active protease bands. After exposure to sodium dodecyl sulfate during gel separation, the proenzymes that were present in the tissue extracts were activated without proteolytic cleavage.

Behavioral testing

The BBB (Basso, Bresnahan, and Beattie) open field score (Basso et al., 1995) was used to evaluate locomotion in terms of hindlimb functional improvement in rats with SCI. The BBB test was scored from 0 (no observable hindlimb movement) to 21 (normal hindlimb movement) points. In this study, behavioral analyses were conducted weekly for 8 weeks postoperation. The behavioral tests were recorded using a video camera and scored by two examiners who were blinded to each behavior evaluation group.

Statistical analysis

Data are expressed as mean ± SEM. Statistical comparisons were performed with GraphPad Prism software (version 7.0, GraphPad Software Inc., La Jolla, CA, USA) using a two-tailed Student's t-test for comparisons of two groups. For comparisons of three or more groups, one-way analysis of variance was performed, combined with Newman-Keuls multiple comparison tests for post hoc analysis. A P-value < 0.05 was used to indicate statistically significant differences between groups.


  Results Top


MMP-2/9 was upregulated post-injury, and MMP-9 was further increased in the graft site

MMP-9 mRNA expression was acutely elevated in group T compared with uninjured rats 2 days following transection but quickly decreased after 6 days [Figure 1]A. These data agreed with the results of a previous report (Noble et al., 2002) wherein upregulation was measured using zymography within 24 hours postinjury, before levels were downregulated at 72 hours postinjury. The levels of MMP-9 mRNA were further increased in the graft site in group R compared with those in group T (6, 10, and 14 days) [Figure 1]A.

MMP-2 was previously reported to be upregulated 5 days after SCI in mice (Dang et al., 2008). Our data demonstrated that MMP-2 mRNA was elevated at 2 days postoperation and further upregulated until 21 days postoperation (final measurement) [Figure 1]B. MMP-2 mRNA was not significantly elevated in group R compared with that in group T. These data suggest that MMP-2/9 were upregulated postinjury and that MMP-9 was further increased in the graft site in group R.

Local inhibition of MMP-2/9 activity was associated with the presence of Arg1+ macrophages in the nerve grafts of repaired spinal cords in rats

To test whether MMP-2/9 inhibition altered the M2 macrophage response, we examined the M1 and M2 macrophage populations. Usually, a combination of markers can allow identification of a population of bona fide ‘type II’ macrophages. In our in situ study, Arg1 was chosen as the M2 macrophage marker, and iNOS was chosen as the M1 marker. Although the data obtained cannot be interpreted as changes in specific, homogenous populations, it is still possible to make some observations regarding changes in macrophage populations. The general MMP inhibitor GM6001 was mixed with fibrin glue and applied to the nerve grafts (group RG), and Arg1 and iNOS gene expression (used as M1 marker) levels were compared using qPCR. The fold change relative to the gene expression in transections with glue (group U) is demonstrated on day 10 postinjury. Arg1 expression was significantly reduced on day 10 with GM6001 application in group RG) compared to that in group R ([Figure 2]A, 10 R vs. 10 RG, P < 0.001), whereas the expression of iNOS was not significantly affected [Figure 2]A. This finding suggests that the MMP inhibitor reduced the M2 macrophage population but did not affect the M1 macrophage population.
Figure 2: Blockage of matrix metalloproteinase (MMP) activity decreased the presence of M2 macrophages in grafted nerves at 10 days postoperation.
ED1 is a monoclonal antibody against the rat CD68 protein and detect activated macrophage. In group T, rats underwent complete transection operation and T8 spinal cord was removed. In group R, spinal cord transected rats received treatment with fibroblast growth factor-1 and peripheral nerve grafts. In group RG, rats received the same treatment as group R with the addition of 200 μM GM6001 (an MMP inhibitor) to the fibrin mix. (A) Levels of gene expression were compared using quantitative polymerase chain reaction. The Y axis is fold change relative to the gene expression of group U (transection plus glue only). On day 10 postoperation, the rats in group R demonstrated a significant difference in Arg1 gene expression compared to the rats in the group U. This elevation was reduced when MMP activity was blocked (10 RG vs. 10R). Left, n = 3, 4, 4; mean ± SEM. Expression of inducible nitric oxide synthase (iNOS) was not significantly changed among groups U, R and RG (right) (mean ± SEM). *P < 0.05, **P < 0.01. (B) Left: Immunofluorescent images of cross sections of grafted nerves double stained with arginase-1 (Arg1) and ED1. The number of Arg1+ macrophages was decreased in the GM6001-treated group (RG) compared with that in the R group (R), whereas the number of ED1+ macrophages was not significantly different between groups R and RG. Right: Analysis of immunoreactivity in the graft area on day 10 postoperation (n = 3, mean ± SEM, *P < 0.05). (C) Left: Western blot analysis of Arg1 and ED1 protein levels in tissue collected from the grafted nerves. Y axis is the arbituary unit from the densitometry for analysing the blot. Right: quantitative analysis (Arg1, n = 5; ED1, n = 4). *P < 0.05, ***P < 0.001.


Click here to view


Immunohistological analysis demonstrated a significant reduction in M2 marker Arg1 immunoreactivity in the graft area when MMP was inhibited on day 10 postoperation (10 R vs. 10 RG, P < 0.05), whereas immunoreactivity for ED1 was not significantly affected [Figure 2]B. ED1 is marker for both M1 and M2 macrophages. The lack of change in ED1 levels demonstrates that the total macrophage population was not significantly reduced and that the Arg1 reduction resulted from changes to a specific population of macrophages. Western blot analysis of the graft area showed a significant reduction in Arg1 levels at the nerve graft site in group RG compared to that in group R at 10 and 14 days postoperation ([Figure 2]C, P < 0.05 and P < 0.001, respectively)

Local inhibition of MMP-2/9 activity impedes BDNF expression and functional recovery

Following SCI, the expression of BDNF mRNA was reported to dramatically be increased within microglia, macrophages and astrocytes at the injury site (Dougherty et al., 2000). It has been previously reported that M2, but not M1, macrophages express BDNF in a myelopathy mouse model with severe compression of the spinal cord (Hirai et al., 2013). We previously reported that the M2 macrophages in the group R expressed more BDNF in the grafted area at 14 days postoperation (Kuo et al., 2011). In the present experiment, we demonstrated that the mRNA expression of BDNF was decreased in the grafted area at 14 days postoperation in group RG relative to that in group R ([Figure 3], 14 RG vs. 14 R, P < 0.05). Therefore, BDNF downregulation was associated with reduced expression of the M2 marker Arg1 when treated with the MMP inhibitor.
Figure 3: Brain-derived neurotrophy factor (BDNF) was decreased in the repaired spinal cords (R group) following treatment with matrix metalloproteinase (MMP) inhibitor GM6001.
BDNF gene expression level was compared using quantitative polymerase chain reaction. The Y axis is fold change relative to the BDNF gene expression of group T (transection plus glue only). In group T, rats underwent complete transection operation and T8 spinal cord was removed. In group R, spinal cord transected rats received treatment with fibroblast growth factor-1 and peripheral nerve grafts. In group RG, rats received the same treatment as group R with the addition of 200 μM GM6001 (an MMP inhibitor) to the fibrin mix. BDNF was increased in the graft areas in group R compared with group T, but was reduced in group RG at 14 days postoperation. One-way analysis of variance combined with Newman-Keuls multiple comparison tests for post hoc analysis was used (mean ± SEM; n = 4; *P < 0.05).


Click here to view


In situ zymography demonstrated that MMP-2/9 activity was detected at 10 and 14 days postoperation in group R but not in group RG [Figure 4]A. Both the pro and active forms of the MMPs were inhibited [Figure 4]B. Hindlimb locomotor function was tested using the BBB score on a weekly basis. At 8 weeks postoperation, the locomotor score of group RG was significantly lower than that of group R and was not significantly different from that of group T ([Figure 4]C, P < 0.05).
Figure 4: Local blockage of matrix metalloproteinase (MMP)-2/9 activity impeded functional recovery of spinal cord rats.
In group T, rats underwent complete transection operation and T8 spinal cord was removed. In group R, spinal cord-transected rats received treatment with fibroblast growth factor-1 and peripheral nerve grafts. In group RG, rats received the same treatment as group R with the addition of 200 μM GM6001 (an MMP inhibitor) to the fibrin mix. (A) In situ zymography demonstrated that MMP-2/9 activity (white signal in photograph) in the graft area of the repaired spinal cord (R), was inhibited by GM6001 (RG) at 10 days postoperation. Scale bars: 1 mm. (B) MMP-2/9 activity in the graft area of the repaired spinal cord (R) at 10 and 14 days (10R, 14R) was reduced compared to group RG (10RG, 14RG), as demonstrated by zymography. (C) The locomotor recovery was evaluated over an 8-week period, using a 21-point scale (Basso, Beattie, Bresnahan (BBB) locomotor rating scale), in groups R, T, and RG. BBB scores were increased in group R (R vs. T, 8 weeks), whereas in group RG it was significantly lower than that in group R and was not different from that in group T (RG vs. T, 8 weeks). One-way analysis of variance combined with Newman-Keuls multiple comparison tests for post hoc analysis was used (mean ± SEM, n = 4, *P < 0.05).


Click here to view



  Discussion Top


Usually with combination of markers could one pinpoint a population that was bona fide ‘type II’ macrophage. In our in situ study Arg1 was chosen for the M2 macrophage and iNOS for M1. The data obtained cannot be interpreted as the activities for the specific populations, but rather, it approaches the reality to a certain degree that we found useful. In relevant places the M1 or M2 macrophage were used instead of Arg1+ or iNOS+ macrophage for a purpose of interpretation and the readers should be aware of the lack of multiple staining for these populations (Shechter et al., 2009, 2013). We previously demonstrated that a combination of peripheral grafts and FGF-1 treatment increased the M2 macrophage population and BDNF expression to promote functional recovery in spinal cord-transected rats (Kuo et al., 2011). In this study, we demonstrated that the expression of the M2 macrophage marker Arg1 was reduced by the MMP inhibitor in the grafted area. The expression of iNOS, an M1 macrophage marker, or ED1, a general marker for activated macrophages, was not significantly reduced, suggesting that the M2 population was preferentially reduced by the MMP inhibitor. At the same time, reduced BDNF expression and a loss of functional recovery were associated with the reduced Arg1 population.

MMP inhibition affects the Arg1+ population significantly possibly because the M2 population secretes more MMP-9 and is more mobile than the M1 population

It is a generally accepted that MMP-9, not MMP-2, supports the migration and infiltration of macrophages into the injured spinal cord (Zhang et al., 2011). A discrepancy exists regarding the expression levels of MMP-9 among the M1 and M2 populations. It has been reported that IL-10-induced M2 macrophages have higher MMP-9 activity and mRNA levels than M1 macrophages in vitro (Chizzolini et al., 2000; Shimizu et al., 2004; Sica et al., 2006; Lolmede et al., 2009; Huang et al., 2012). In addition, as reported, M1 macrophages are relatively immobile, whereas M2 macrophages are more mobile (Lively and Schlichter, 2013). It is known that stem cell transplantation-induced M2 macrophage migration is accompanied by the upregulation of MMP-9 (Pastrana et al., 2006; Ding et al., 2009; Veeravalli et al., 2009). Human mesenchymal stem cell treatment significantly increased MMP-9 activity and M2 macrophage gene expression in kidneys with ischemia-reperfusion injury in mice (Wise et al., 2014). This evidence may explain the more active migration of the M2 population of macrophages. If the Arg1+ population represents most of the M2 population, it follows that the more mobile population was affected when MMPs were inhibited in this study.

MMP inhibition may have differentially blocked the migration of a remote, endogenous M2 population after SCI

It has been recently discovered that the migration of M1 and M2 macrophages is distinctly regulated via different pathways in contusive SCI. Notably, M2 macrophages differentiate from monocytes, which are trafficked through the remote brain ventricular choroid plexus (CP) to reach the location of the injury (Shechter et al., 2013). In addition, the migration of the M1 and M2 populations can be regulated locally near the injury penumbra and is involved in the modulation of the ECM, which is an MMP target (Shechter et al., 2011; Vadivelu et al., 2015). Therefore, homing of M1 and M2 populations to the injury site is differentially regulated both chronologically and geographically. If the Arg1+ population we observed in the nerve graft represents most of the incoming M2 population, our results indicate that some of the determinants of M2 entry into the injury site were locally distributed and may be regulated by MMPs. This result suggests that the Arg1+ population, or at least some of the M2 population in our ‘repair’ model, may be a separate migratory cell population rather than the phenotypic conversion of early M1 cells.

MMP9 was associated with the M2 population and created a permissive environment for axonal regeneration

Recently, the M2-associated protein decoy receptor 3 has been observed to induce macrophages to secret more MMP-9 than IL-4-induced macrophages (Chang et al., 2008), to increase the population of M2 macrophages in the lesioned area and to promote functional recovery in contusive SCI (Chiu et al., 2016). In this study, we found that MMP-9 expression was increased and was associated primarily with macrophages in the graft area of the repaired spinal cord, where Arg1+ macrophages were also increased compared to the transected spinal cord. In addition, we observed that the MMP inhibitor in the grafted area reduced Arg1+ (M2 marker) gene expression, whereas iNOS (M1 marker) and ED1 (activated macrophages) expression was not significantly affected. This result suggests that MMP-9 may be associated with the M2 macrophage population in the grafted area, thereby creating a more permissive environment for functional recovery in our repaired model.

An increased M2 population was associated with many beneficial effects for recovery from SCI and should not be compromised

Several strategies, including cell therapy, molecular therapy, and combinatorial treatment, have been developed in SCI animal models (Chang et al., 2006; Ahn et al., 2012; Silva et al., 2014). Increasing evidence demonstrates that combinatorial strategies employing multiple agents, such as pertinent neurotrophic factors, scar-resolving agents, or other axonal growth-promoting agents, are able to further improve locomotor functions after SCI (Didangelos et al., 2014). In our previous study, we demonstrated that local BDNF was increased on day 14 postoperation in the repaired spinal cord of rats (Kuo et al., 2011). In this study, we demonstrated that reduction of an Arg1+ population by the MMP inhibitor was associated with BDNF reduction in grafted nerves and worse functional recovery. This result indicates that increased Arg1+ cells, or at least some M2 macrophages, were associated with multiple beneficial effects. MMPs themselves have been considered a drug target for the promotion of CNS repair; however, if the mechanism of repair involves the elevation of M2 macrophage activity, treatment with an MMP inhibitor may compromise macrophage activity and should be used with caution.

Altogether, this study indicates that MMPs may contribute to the therapeutic features of M2 macrophages through the control of their motility in our repair model, as well as through providing a more permissive environment. The M2 population in group R may be a migratory population rather than the phenotypic conversion of early M1 macrophages. Application of the MMP inhibitor and the promotion of MMP activity require more consideration and investigation of the mechanisms of the coadministered treatments.

About the ProCord clinical trial and our treatment

The ProCord clinical trial utilized the infusion of autologous macrophages for the treatment of completely transected spinal cord patients. Although the initial proof of safety was provided in 2005 for the phase I trial and several cases progress from AISA A to ASIA C (Knoller et al., 2005), the randomized controlled multicenter trial in phase II failed to show a significant difference in primary outcomes between the treated and control groups (Lammertse et al., 2012). Our group R treatment that resulted in vast Arg1+ macrophage accumulation in the injury site could be similar to the peripheral nerve preincubated macrophage injection. However, the distribution and activities still differ. Further, the lack of details for the proprietary treatment of the clinical trials make it difficult for us to say whether the procedure would be different from our peripheral nerve grafts plus FGF-1 treatment. As one of the authors who published the result demonstrating behavioral improvement with activated macrophage treatment stated, the type of macrophages and the time and location of their activity would influence regeneration (Shechter and Schwartz, 2013). Our data suggest that macrophage activity for spinal regeneration remains an interesting topic.[52]

Acknowledgments: The authors thank Eusol Biotech Co. Ltd., for providing FGF-1. We thank Dr. Dinesh Chandra Agrawal from Department of Applied Chemistry, Chaoyang University of Technology, for writing assistance. We also thank the Medical Science & Technology Building of Taipei Veterans General Hospital, China for providing an experimental space and facilities.

Author contributions: CWC were involved in the data acquisition, data analysis, and writing of part of the manuscript. CJC performed both data acquisition and analysis. HSK and MJT were involved in the data interpretation. WHH drafted some part of manuscript. MJL and HC participated in the study design and conceptualization and draft part of the manuscript. All authors read and approved the final manuscript.

Conflicts of interest: The authors declare no conflicts of interest.

Financial support: This work was supported by the National Science Council (102-2320-B-324-001), China. Other funding was provided by grants from Taipei Veterans General Hospital (V103E6-001 & V104E6-001) and by grants (MOST 104-2314-B-010-012-MY3, MOST 105-2314-B-010-013-MY2 and MOST 106-2632-B-324-001) from the Ministry of Science and Technology in Taiwan, China.

Institutional review board statement: This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Veterans General Hospital, China (approval number IACUC 2015-167).

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Open peer reviewers: Gregory Hawryluk, The University of Utah, USA; Viness Pillay, University of the Witwatersrand, France; Brent Neumann, Monash University, Faculty of Medicine, Nursing and Health Sciences, Australia.

Additional file: Open peer review reports 1, 2 and 3[Additional file 1].

Funding: This work was supported by the National Science Council (102-2320-B-324-001), China. This study was also supported by grants from Taipei Veterans General Hospital (V103E6-001 & V104E6-001) and by grants (MOST 104-2314-B-010-012-MY3, MOST 105-2314-B-010-013-MY2 and MOST 106-2632-B-324-001) from the Ministry of Science and Technology in Taiwan, China.



 
  References Top

1.
Ahn M, Lee C, Jung K, Kim H, Moon C, Sim KB, Shin T (2012) Immunohistochemical study of arginase-1 in the spinal cords of rats with clip compression injury. Brain Res 1445:11-19.  Back to cited text no. 1
    
2.
Bartus K, James ND, Didangelos A, Bosch KD, Verhaagen J, Yanez-Munoz RJ, Rogers JH, Schneider BL, Muir EM, Bradbury EJ (2014) Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury. J Neurosci 34:4822-4836.  Back to cited text no. 2
    
3.
Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1-21.  Back to cited text no. 3
    
4.
Busch SA, Horn KP, Silver DJ, Silver J (2009) Overcoming macrophage-mediated axonal dieback following CNS injury. J Neurosci 29:9967-9976.  Back to cited text no. 4
    
5.
Chang YC, Chan YH, Jackson DG, Hsieh SL (2006) The glycosaminoglycan-binding domain of decoy receptor 3 is essential for induction of monocyte adhesion. J Immunol 176:173-180.  Back to cited text no. 5
    
6.
Chang YC, Chen TC, Lee CT, Yang CY, Wang HW, Wang CC, Hsieh SL (2008) Epigenetic control of MHC class II expression in tumor-associated macrophages by decoy receptor 3. Blood 111:5054-5063.  Back to cited text no. 6
    
7.
Cheng H, Cao Y, Olson L (1996) Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function. Science 273:510-513.  Back to cited text no. 7
    
8.
Cherry JD, Olschowka JA, O’Banion MK (2014) Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation 11:98.  Back to cited text no. 8
    
9.
Chiu CW, Huang WH, Lin SJ, Tsai MJ, Ma H, Hsieh SL, Cheng H (2016) The immunomodulator decoy receptor 3 improves locomotor functional recovery after spinal cord injury. J Neuroinflammation 13:154.  Back to cited text no. 9
    
10.
Chizzolini C, Rezzonico R, De Luca C, Burger D, Dayer JM (2000) Th2 cell membrane factors in association with IL-4 enhance matrix metalloproteinase-1 (MMP-1) while decreasing MMP-9 production by granulocyte-macrophage colony-stimulating factor-differentiated human monocytes. J Immunol 164:5952-5960.  Back to cited text no. 10
    
11.
Dang AB, Tay BK, Kim HT, Nauth A, Alfonso-Jaume MA, Lovett DH (2008) Inhibition of MMP2/MMP9 after spinal cord trauma reduces apoptosis. Spine 33:E576-579.  Back to cited text no. 11
    
12.
David S, Kroner A (2011) Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci 12:388-399.  Back to cited text no. 12
    
13.
Didangelos A, Iberl M, Vinsland E, Bartus K, Bradbury EJ (2014) Regulation of IL-10 by chondroitinase ABC promotes a distinct immune response following spinal cord injury. J Neurosci 34:16424-16432.  Back to cited text no. 13
    
14.
Ding Y, Xu D, Feng G, Bushell A, Muschel RJ, Wood KJ (2009) Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9. Diabetes 58:1797-1806.  Back to cited text no. 14
    
15.
Dougherty KD, Dreyfus CF, Black IB (2000) Brain-derived neurotrophic factor in astrocytes, oligodendrocytes, and microglia/macrophages after spinal cord injury. Neurobiol Dis 7:574-585.  Back to cited text no. 15
    
16.
Franco R, Fernandez-Suarez D (2015) Alternatively activated microglia and macrophages in the central nervous system. Prog Neurobiol 131:65-86.  Back to cited text no. 16
    
17.
Gensel JC, Zhang B (2015) Macrophage activation and its role in repair and pathology after spinal cord injury. Brain Res 1619:1-11.  Back to cited text no. 17
    
18.
Ghannam S, Bouffi C, Djouad F, Jorgensen C, Noel D (2010) Immunosuppression by mesenchymal stem cells: mechanisms and clinical applications. Stem Cell Res Ther 1:2.  Back to cited text no. 18
    
19.
Gong Y, Hart E, Shchurin A, Hoover-Plow J (2008) Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest 118:3012-3024.  Back to cited text no. 19
    
20.
Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3:23-35.  Back to cited text no. 20
    
21.
Hirai T, Uchida K, Nakajima H, Guerrero AR, Takeura N, Watanabe S, Sugita D, Yoshida A, Johnson WE, Baba H (2013) The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinal cord compression: implications for human cervical compressive myelopathy. PLoS One 8:e64528.  Back to cited text no. 21
    
22.
Hu X, Leak RK, Shi Y, Suenaga J, Gao Y, Zheng P, Chen J (2015) Microglial and macrophage polarization-new prospects for brain repair. Nat Rev Neurol 11:56-64.  Back to cited text no. 22
    
23.
Huang WC, Sala-Newby GB, Susana A, Johnson JL, Newby AC (2012) Classical macrophage activation up-regulates several matrix metalloproteinases through mitogen activated protein kinases and nuclear factor-kappaB. PLoS One 7:e42507.  Back to cited text no. 23
    
24.
Jesnowski R, Backhaus C, Ringel J, Lohr M (2002) Ribosomal highly basic 23-kDa protein as a reliable standard for gene expression analysis. Pancreatology 2:421-424.  Back to cited text no. 24
    
25.
Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29:13435-13444.  Back to cited text no. 25
    
26.
Knoller N, Auerbach G, Fulga V, Zelig G, Attias J, Bakimer R, Marder JB, Yoles E, Belkin M, Schwartz M, Hadani M (2005) Clinical experience using incubated autologous macrophages as a treatment for complete spinal cord injury: phase I study results. J Neurosurg Spine 3:173-181.  Back to cited text no. 26
    
27.
Kuo HS, Tsai MJ, Huang MC, Chiu CW, Tsai CY, Lee MJ, Huang WC, Lin YL, Kuo WC, Cheng H (2011) Acid fibroblast growth factor and peripheral nerve grafts regulate Th2 cytokine expression, macrophage activation, polyamine synthesis, and neurotrophin expression in transected rat spinal cords. J Neurosci 31:4137-4147.  Back to cited text no. 27
    
28.
Kuo HS, Tsai MJ, Huang MC, Huang WC, Lee MJ, Kuo WC, You LH, Szeto KC, Tsai IL, Chang WC, Chiu CW, Ma H, Chak KF, Cheng H (2007) The combination of peripheral nerve grafts and acidic fibroblast growth factor enhances arginase I and polyamine spermine expression in transected rat spinal cords. Biochem Biophys Res Commun 357:1-7.  Back to cited text no. 28
    
29.
Lammertse DP, Jones LA, Charlifue SB, Kirshblum SC, Apple DF, Ragnarsson KT, Falci SP, Heary RF, Choudhri TF, Jenkins AL, Betz RR, Poonian D, Cuthbert JP, Jha A, Snyder DA, Knoller N (2012) Autologous incubated macrophage therapy in acute, complete spinal cord injury: results of the phase 2 randomized controlled multicenter trial. Spinal Cord 50:661-671.  Back to cited text no. 29
    
30.
Lee MJ, Chen CJ, Huang WC, Huang MC, Chang WC, Kuo HS, Tsai MJ, Lin YL, Cheng H (2011) Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1. Neuropathol Appl Neurobiol 37:585-599.  Back to cited text no. 30
    
31.
Lee YS, Hsiao I, Lin VW (2002) Peripheral nerve grafts and aFGF restore partial hindlimb function in adult paraplegic rats. J Neurotrauma 19:1203-1216.  Back to cited text no. 31
    
32.
Lee YS, Lin CY, Robertson RT, Hsiao I, Lin VW (2004) Motor recovery and anatomical evidence of axonal regrowth in spinal cord-repaired adult rats. J Neuropathol Exp Neurol 63:233-245.  Back to cited text no. 32
    
33.
Letellier E, Kumar S, Sancho-Martinez I, Krauth S, Funke-Kaiser A, Laudenklos S, Konecki K, Klussmann S, Corsini NS, Kleber S, Drost N, Neumann A, Lévi-Strauss M, Brors B, Gretz N, Edler L, Fischer C, Hill O, Thiemann M, Biglari B, et al. (2010) CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site. Immunity 32:240-252.  Back to cited text no. 33
    
34.
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.  Back to cited text no. 34
    
35.
Lively S, Schlichter LC (2013) The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasion. J Neuroinflammation 10:75.  Back to cited text no. 35
    
36.
Lolmede K, Campana L, Vezzoli M, Bosurgi L, Tonlorenzi R, Clementi E, Bianchi ME, Cossu G, Manfredi AA, Brunelli S, Rovere-Querini P (2009) Inflammatory and alternatively activated human macrophages attract vessel-associated stem cells, relying on separate HMGB1- and MMP-9-dependent pathways. J Leukoc Biol 85:779-787.  Back to cited text no. 36
    
37.
Mantovani A, Biswas SK, Galdiero MR, Sica A, Locati M (2013) Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol 229:176-185.  Back to cited text no. 37
    
38.
Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25:677-686.  Back to cited text no. 38
    
39.
Noble LJ, Donovan F, Igarashi T, Goussev S, Werb Z (2002) Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events. J Neurosci 22:7526-7535.  Back to cited text no. 39
    
40.
Pastrana E, Moreno-Flores MT, Gurzov EN, Avila J, Wandosell F, Diaz-Nido J (2006) Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. J Neurosci 26:5347-5359.  Back to cited text no. 40
    
41.
Shechter R, Schwartz M (2013) Harnessing monocyte-derived macrophages to control central nervous system pathologies: no longer ‘if’ but ‘how’. J Pathol 229:332-346.  Back to cited text no. 41
    
42.
Shechter R, Raposo C, London A, Sagi I, Schwartz M (2011) The glial scar-monocyte interplay: a pivotal resolution phase in spinal cord repair. PLoS One 6:e27969.  Back to cited text no. 42
    
43.
Shechter R, London A, Varol C, Raposo C, Cusimano M, Yovel G, Rolls A, Mack M, Pluchino S, Martino G, Jung S, Schwartz M (2009) Infiltrating blood-derived macrophages are vital cells playing an anti-inflammatory role in recovery from spinal cord injury in mice. PLoS Med 6:e1000113.  Back to cited text no. 43
    
44.
Shechter R, Miller O, Yovel G, Rosenzweig N, London A, Ruckh J, Kim KW, Klein E, Kalchenko V, Bendel P, Lira SA, Jung S, Schwartz M (2013) Recruitment of beneficial M2 macrophages to injured spinal cord is orchestrated by remote brain choroid plexus. Immunity 38:555-569.  Back to cited text no. 44
    
45.
Shimizu K, Shichiri M, Libby P, Lee RT, Mitchell RN (2004) Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest 114:300-308.  Back to cited text no. 45
    
46.
Sica A, Schioppa T, Mantovani A, Allavena P (2006) Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer 42:717-727.  Back to cited text no. 46
    
47.
Silva NA, Sousa N, Reis RL, Salgado AJ (2014) From basics to clinical: a comprehensive review on spinal cord injury. Prog Neurobiol 114:25-57.  Back to cited text no. 47
    
48.
Tian YF, Zhang PB, Xiao XL, Zhang JS, Zhao JJ, Kang QY, Chen XL, Qiu F, Liu Y (2007) The quantification of ADAMTS expression in an animal model of cerebral ischemia using real-time PCR. Acta Anaesthesiol Scand 51:158-164.  Back to cited text no. 48
    
49.
Vadivelu S, Stewart TJ, Qu Y, Horn K, Liu S, Li Q, Silver J, McDonald JW (2015) NG2+ progenitors derived from embryonic stem cells penetrate glial scar and promote axonal outgrowth into white matter after spinal cord injury. Stem Cells Transl Med. 4:401-411.  Back to cited text no. 49
    
50.
Veeravalli KK, Dasari VR, Tsung AJ, Dinh DH, Gujrati M, Fassett D, Rao JS (2009) Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury. Neurobiol Dis 36:200-212.  Back to cited text no. 50
    
51.
Wise AF, Williams TM, Kiewiet MB, Payne NL, Siatskas C, Samuel CS, Ricardo SD (2014) Human mesenchymal stem cells alter macrophage phenotype and promote regeneration via homing to the kidney following ischemia-reperfusion injury. Am J Physiol Renal Physiol 306:F1222-1235.  Back to cited text no. 51
    
52.
Zhang H, Trivedi A, Lee JU, Lohela M, Lee SM, Fandel TM, Werb Z, Noble-Haeusslein LJ (2011) Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood-borne monocytes into the injured spinal cord. J Neurosci 31:15894-15903.  Back to cited text no. 52
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1]


This article has been cited by
1 TAT-modified tetramethylpyrazine-loaded nanoparticles for targeted treatment of spinal cord injury
Jian Li,Jun Wei,Yujie Wan,Xingjie Du,Xiaosheng Bai,Chunhong Li,Yan Lin,Zhongbing Liu,Meiling Zhou,Zhirong Zhong
Journal of Controlled Release. 2021;
[Pubmed] | [DOI]
2 Application of fibrin-based hydrogels for nerve protection and regeneration after spinal cord injury
Ziyuan Yu,Hongru Li,Peng Xia,Weijian Kong,Yuxin Chang,Chuan Fu,Kai Wang,Xiaoyu Yang,Zhiping Qi
Journal of Biological Engineering. 2020; 14(1)
[Pubmed] | [DOI]
3 Role of macrophages in peripheral nerve injury and repair
Ping Liu,Jiang Peng,Gong-Hai Han,Xiao Ding,Shuai Wei,Gang Gao,Kun Huang,Feng Chang,Yu Wang
Neural Regeneration Research. 2019; 14(8): 1335
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  Materials and Me...
  In this article
Abstract
Introduction
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed2261    
    Printed19    
    Emailed0    
    PDF Downloaded244    
    Comments [Add]    
    Cited by others 3    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]