LINGO-1 deficiency promotes nerve regeneration through reduction of cell apoptosis, inflammation, and glial scar after spinal cord injury in mice

Leucine-rich repeat and immunoglobulin domain-containing protein 1 (LINGO-1) is a transmembrane protein that negatively regulates neural regeneration in the central nervous system. LINGO-1 expression is up-regulated after central nerve injury, and is accompanied by cell death. Both LINGO-1 and cell death in the injury microenvironment are thought to limit neural regeneration, but the relationship between LINGO-1 and cell death has not been characterized. To investigate whether LINGO-1 deletion improves the spinal cord microenvironment after spinal cord injury (SCI) and contributes to cell survival, we generated LINGO-1 knockout (KO) mice. These mice and wild-type control mice were subjected to spinal cord transection. Fourteen days after spinal cord transection, cell apoptosis, inflammation, glial scar, and growth of nerve fibers were evaluated by immunostaining. The results showed that LINGO-1 KO mice demonstrated a profound reduction in expression of caspase-3, transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL), ionized calcium binding adapter molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and chondroitin sulfate proteoglycans (CSPGs) compared to controls. In contrast, expression of neurofilament (NF) at the SCI site in LINGO-1 KO mice was markedly increased compared to that in wild-type mice. These results suggested that LINGO-1 plays a critical role in the injury microenvironment in processes such as cell death, inflammatory response, and glial scar formation. Importantly, LINGO-1 deletion and a positive microenvironment may exert synergistic effects to promote nerve fiber regeneration. Therefore, inhibition of LINGO-1 may be a therapeutic strategy to promote neural regeneration following SCI.


Introduction
Spinal cord injury (SCI) typically includes an initial injury and a secondary injury. In the primary injury, red blood cells, myelin, subcellular debris, and necrotic and apoptotic neurons damaged by the traumatic injury are featured prominently in the lesion epicenter, leading to secondary injury (Tran et al., 2018). Secondary injury mainly includes inflammation, extravasation of infiltrating leukocytes, and proliferation and morphological changes in glial cells (Tran et al., release large amounts of alarmins such as chromatin-associated proteins (high mobility group box-1 protein, HMGB1), adenosine triphosphate (ATP), histones, S100 (a family of low-molecular-weight proteins), and interleukins, which can initiate a cascade of inflammatory signaling (Bianchi, 2007;Kono and Rock, 2008;Tran et al., 2018). Resident glial cells, including astrocytes, oligodendrocytes, and microglia, respond swiftly to the altered microenvironment (Tran et al., 2018). Reactive astrocytes undergo proliferation and migrate into the lesion site, forming astroglial scars, which are mechanical barriers to axonal regeneration (Losey et al., 2014;Rhodes and Fawcett, 2004;Yiu and He, 2006). Moreover, in the inflammatory environment, reactive astrocytes increase secretion of chondroitin sulfate proteoglycans (CSPGs), which inhibit axon outgrowth (David et al., 2012;McKeon et al., 1991;Ughrin et al., 2003;Yiu and He, 2006), thus creating a chemical barrier (Losey et al., 2014;Yiu and He, 2006). Therefore, astrocytic glial scars created by chronic secondary injury result in inhibition of axonal growth (Tran et al., 2018).
Leucine-rich repeat and immunoglobulin domain-containing protein (LINGO-1) is a member of the LIG gene super-family, a family of type I membrane proteins containing extracellular domains composed of C2 immunoglobulin-like domains and leucine-rich repeats (Meabon et al., 2016). LINGO-1 is abundantly and exclusively expressed in the central nervous system (Carim-Todd et al., 2003). It is a potent negative regulator of neuron and oligodendrocyte survival, axon extension, axon regeneration, oligodendrocyte differentiation, and axonal myelination (Andrews and Fernandez-Enright, 2015). LINGO-1 has been widely studied in multiple sclerosis (MS) and SCI due to its remarkable role in neurite outgrowth, oligodendrocyte differentiation, and myelination (Ji et al., 2006;Mi et al., 2007). A previous study showed that LINGO-1 level rose 14 days after SCI (Foale et al., 2017;Mi et al., 2004), and inhibition of LINGO-1 increased axonal sprouting, and neuronal and oligodendrocyte survival due to inhibition of RhoA activation (Ji et al., 2006). Mi et al. observed a marked increase in myelinated axons within the spinal cords of LINGO-1 knockout (KO) mice compared to wild-type (WT) mice in an MS model . Administration of a LINGO-1 antagonist and LINGO-1-Fc antibody resulted in improved functional recovery of rats following SCI. A previous study showed that LINGO-1 KO mice had a higher percentage of mature oligodendrocytes and earlier appearance of axonal myelination in the central nervous system than WT mice in an MS model Mi et al., 2005). Moreover, compared with WT mice, increased survival of dopaminergic neurons was observed in LINGO-1 KO mice in a Parkinson's disease model (Inoue et al., 2007).
While the role of LINGO-1 in axonal growth and oligodendrocyte maturation has been well characterized, its relationship with the inhibitory microenvironment during pathological conditions, such as complete spinal cord transection injury, has not been evaluated. It is widely accepted that LINGO-1 is a negative regulator of cell survival and nerve regeneration, and a microenvironment enriched in LINGO-1 in injured spinal cord can inhibit cell survival and nerve regeneration. The extent to which LINGO-1 KO reduces cell apoptosis, improves the spinal cord microenvironment, and promotes nerve regeneration in the SCI site following complete transection remains to be determined.
SCI models can be classified as contusion, compression, distraction, dislocation, transection, and chemical models. They are chosen depending on the study goals, characteristics of the model, and study resources (Cheriyan et al., 2014). Complete transection is easy to perform, and results in a complex pathophysiological cascade that inhibits potential sprouting from spared axons and leads to formation of scar tissue (Cheriyan et al., 2014;Li et al., 2019). Complete transection is usually regarded as the gold standard for evaluation of nerve fiber regeneration in the SCI site . Therefore, complete transection with evaluation of nerve regeneration, apoptosis, inflammation, and glial scar formation was selected for investigation of the effects of LINGO-1.
In this study, we used CRISPR/Cas9 technology to generate LINGO-1 KO mice. These mice, and WT control mice, underwent complete spinal cord transection. Fourteen days after spinal cord transection, the number of apoptotic cells was lower in LINGO-1 KO mice than in WT mice. In addition, we observed lower expression of ionized calcium binding adapter molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and CSPGs in and around the SCI site in LINGO-1 KO mice compared with WT mice. Our study suggested that LINGO-1 KO increased regeneration of nerve fibers in the SCI site through decreased cell apoptosis and improvement of the spinal cord microenvironment, which included attenuation of inflammation and glial scar formation.

Animals
Healthy C57BL/6J female mice (21-28 days old) were selected as oocyte donors for superovulation, and then were mated to C57BL/6J stud male mice (49-56 days old) and fertilized as zygote donors. CD1 male mice (≥56 days old) with good mating records were selected to undergo vasectomies, and then mated to CD1 female mice (≥42 days old) to produce pseudo-pregnant mice, which were used as embryo recipients. All animals were housed at the Laboratory Animal Center of Sun Yat-sen University, which is fully accredited by the Animal Care and Use Committee of Sun Yat-sen University, and all experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Construction of the plasmid co-expressing Cas9 and single-guide RNA (sgRNA)
sgRNAs were designed using the online tools (http://crispr.mit.edu/ ) developed by the Feng Zhang research group and the gRNA with the highest score (score of 95) was chosen for this study. The pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene plasmid #42230, a gift from Feng Zhang) expressing Cas9 and sgRNA (Cong et al., 2013) was digested with BbsI and dephosphorylated using Antarctic Phosphatase (NEB, UK). The linearized vector was purified using QIAquick Gel Extraction Kit (QIAGEN, USA). To generate the bicistronic vector (pX330-Cas9-LINGO-1) expressing Cas9 and sgRNA against LINGO-1 , a pair of oligos for targeting LINGO-1 exon7 (forward: 5′-CAC CGCCGCCACAAAGCGTTTGCGG-3′; reverse: 5′-AAACCCGCAAACGCTT TGTGGCGGC-3′) were annealed, phosphorylated, and ligated to linearize the vector. The ligated vectors were transfected into Stbl3 competent bacteria and inoculated on LB agarose plate. Ten colonies were selected for sequencing analysis to confirm successful ligation of the oligos and the pX330 plasmid. The forward sequence primer was GAGGGCCTATTTCCCATGATT. The successfully ligated clone was amplified for large plasmid yield and extracted using the EndoFree Plasmid Maxi Kit (QIAGEN, USA).

Primary culture of mouse neural stem cells (NSCs)
Cultured neural stem cells were obtained from the hippocampal tissue of C57BL/6 J mice according to a previously published procedure (Zeng et al., 2005). Briefly, 1-3-day-old mice were anesthetized and the hippocampus was dissociated in cold D-Hank's solution. The suspension was centrifuged at 1000 rpm for 5 min and the cell pellet was resuspended in basal medium (Zeng et al., 2005). The NSCs were cultured at 37°C in a 5% CO 2 incubator.

Activity of the pX330-Cas9-LINGO-1 plasmid
After NSCs were cultured for 5-7 days, they were transfected with 800 ng pX330-Cas9-LINGO-1 plasmid and 800 ng pX330 plasmid (negative control) for 48 h using Nucleofector Kit and 4D-Nucleofector (Lonza, Germany) following the manufacturer's protocol. The genomic region surrounding the CRISPR target for LINGO-1 was amplified by PCR using the One Taq DNA Polymerase kit (NEB, USA) according to the manufacturer's instructions. The forward primer was: 5′-TGAACC CTAAAGGAGATGGCACTG-3′, and the reverse primer was: 5′-GTTCTC ACTGATGTCCAGCTTGG-3′. The PCR program was as follows: 95°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, for 35 cycles, then 72°C for 10 min. The PCR products were sequenced to detect genome modification.

Production of LINGO-1 sgRNA
To obtain LINGO-1 sgRNA for microinjection, T7 promoter was (caption on next page) L.-J. Huang, et al. Experimental Neurology 320 (2019) 112965 added to the LINGO-1 sgRNA template by PCR amplification using the following primers. The forward primer was: 5′-TAATACGACTCACTAT AGGGCCGCCACAAAGCGTTTGCGG-3′, and the reverse primer was 5′-AAAAGCACCGACTCGGTGCC-3′. The PCR product was purified using a QIAquick PCR Purification kit (QIAGEN, USA). The T7-LINGO-1 sgRNA purified PCR product was used as the template using the MEGAshortscript T7 kit (ThermoFisher Scientific, USA) for in vitro transcription. Then, the transcript of LINGO-1 sgRNA was purified using the MEGAclear kit (ThermoFisher Scientific, USA) and eluted in RNAsefree water. The quality of LINGO-1 sgRNA was evaluated by agarose gel electrophoresis.

Microinjection into zygotes and embryo transfer
Female C57BL/6J (21-28-day-old) mice were injected with PMSG (5 IU) at 1:00-2:00 p.m. on day 1, injected with hCG (5IU) 48 h later, then housed with male C57BL/6J mice overnight. On the next morning, female mice with plugs were collected for zygote preparation . Zygote-cumulus complexes were collected from the oviducts, separated into single fertilized eggs in M2 medium, and then placed into KSOM medium at 37°C in a 5% CO 2 incubator . LINGO-1 sgRNA (50 ng/μl) and Cas9 mRNA (Biomics Biotechnologies, China) (100 ng/μl) diluted in injection buffer were injected into the cytoplasm of zygotes. The injected zygotes were cultured in KSOM medium at 37°C in a 5% CO 2 incubator until the two-cell stage or the blastocyst stage, then transferred into the uteruses of surrogate female mice, and grown into F0 generation mice .

Identification and genotyping of transgenic mice
Mouse tails were clipped 21 days after birth, and PCR and sequencing were performed to identify and characterize founder (F0) mice. Total DNA was isolated from tails for all mice. PCR and sequencing assay were performed using the protocol described above. When mice with modified genes were identified in the F0 generation, they were selected to interbreed with WT mice. This interbreeding led to F1 generation mice, and they were identified by PCR (forward primer: 5′-TGAACCCTAAAGGAGATGGCACTG-3′, reverse primer: 5′-GTTCTCACT GATGTCCAGCTTGG-3′) and sequencing. When mice with modified genes were identified in this generation, the PCR products of genetically modified mice were analyzed by TA clone experiments. The PCR products were purified by agarose gel electrophoresis and joined with T vector. They were then transfected into DH5α competent cells. The successfully transfected cells were screened using Luria-Bertani (LB) solid medium coated with X-Gal and isopropyl β-D-thiogalactoside (IPTG). Ten white bacterial colonies were selected for sequencing analysis to characterize the modified sequence. Thereafter, male and female heterozygotes with the same modified gene sequence were mated to produce F2 generation mice, and homozygotes were screened out by PCR (primers were the same as those discussed above) and sequencing.

Spinal cord transection
Sixty-four adult female mice (56 days old) were randomly divided into two groups that underwent spinal cord transection: 1. LINGO-1 +/+ group (WT mice, n = 32); 2. LINGO-1 −/− group (LINGO-1 KO homozygote mice, n = 32). All animals were anesthetized with 1% sodium pentobarbital (6.67 μl/g, i.p.). A dorsal laminectomy was performed at the T9 vertebral level to expose the T9 and T10 spinal cord segments. The dura mater, arachnoid, and pia mater were slit open successively, and then the T10 segment of the spinal cord was transected completely with a straight trabecular scissors. The transection site was carefully examined and the wound was sutured after thorough hemostasis. Dysfunction of bladder control was a transient side effect in all mice that underwent spinal cord transection. Manual emptying of the bladders was performed 3 times per day. After surgery, the mice received an intramuscular injection of penicillin (32,000 U/ml/day) within the first 3 days to prevent infection. Both groups of mice were sacrificed 14 days after transection.

Western blot analysis in mice
At 3, 7, and 14 days after spinal cord transection, some of the mice (n = 8 for each group) were deeply anesthetized and sacrificed using 2% sodium pentobarbital (13.34 μl/g, i.p.). A 1-cm segment of the spinal cord extending from T8 to T11 containing the injury site was quickly dissected on ice. Protein from the spinal cord segments was extracted using protein lysis buffer (Huang et al., 2014) and quantified using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of the protein suspension were separated by 12% sodium  Huang, et al. Experimental Neurology 320 (2019) 112965 WBKLS0500, USA). The gray value of bands was analyzed using Image J.

Morphological quantification
To observe the areas 0.5 mm rostral and 0.5 mm caudal to the injury site, as well as the SCI site, every fifth longitudinal section from each spinal cord was evaluated. Twenty-five sections taken from each mouse were immunostained with transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL), APC, MAP2, IBA1, GFAP, CSPGs, and NF antibodies. The stained sections were imaged and analyzed using a TissueFAXS 200 flow-type tissue quantitative analyzer (TissueGnostics GmbH, Vienna, Austria). Expression of the proteins listed above was performed in the injured region, and the areas 0.5 mm rostral and 0.5 mm caudal to the injury site, as demarcated by 4 red lines (Fig. S1). The percentage of apoptotic oligodendrocytes and neurons was obtained by counting APC/TUNEL-and MAP2/TUNEL-positive cells with well-delineated nuclei counterstained with Hoechst33342. The percentage of microglial cells/macrophages was obtained by counting IBA1-positive cells with well-delineated nuclei counterstained with Hoechst33342. We used GFAP and CSPG immunofluorescence staining to identify glial scars, and NF immunoreactive staining to determine the number of NF-positive nerve fibers. The area percentages occupied by the glial scars and nerve fibers were determined from the ratio of the positive immunoreactive area to the total tissue area between the two red lines (Fig. S1).

Statistical analysis
The data were analyzed using Student's t-test and presented as means ± standard deviation (SD). Statistical significance was calculated using the statistical software SPSS 13.0. P < 0.05 was considered statistically significant.

Generation of LINGO-1 −/− mice
To assess the effect of LINGO-1 on SCI, we designed an sgRNA which was cloned into a pX330 plasmid expressing Cas9 and driven by the U6 promoter (Fig. 1A, B, Fig. S2A). PCR products from NSCs transfected with pX330-Cas9-LINGO-1 vector exhibited double peaks in the region corresponding to sgRNA (Fig. S2B, C). The sgRNA band indicated that the sgRNA was successfully obtained (Fig. 1C). The LINGO-1 sgRNA and Cas9 mRNA were co-injected into one-cell embryos (Fig. 1D), and 1 female mouse and 6 male mice were generated for the F0 generation (Table S1, Fig. S3A). TA cloning analysis of genetically modified mice in the F1 generation showed that there were five different mutations in the LINGO-1 gene (Fig. S3B), and homozygous mice with the 23 bp deletion in the F2 generation were obtained (Table S2, Fig. 1E). The central nervous tissue of homozygous mice with the 23 bp deletion was evaluated for LINGO-1 expression by Western blot, and the results showed that there was no band in the 23 bp-LINGO-1 −/− mice compared with the LINGO-1 +/+ mice (Fig. 1F), suggesting that the LINGO-1 −/− mice had been constructed successfully using the CRISPR/Cas9 system. To determine whether there were spinal cord abnormalities in adult LINGO-1 −/− mice, the proportion of gray matter to white matter at C4-C5, T9-10, and L3-L5 was determined by neutral red staining, which showed no obvious differences between LINGO-1 +/+ and LINGO-1 −/− mice (Fig. S4).

Cell apoptosis in the SCI site of LINGO-1 KO mice
We examined the effect of LINGO-1 on cell apoptosis by implementing a T9 and T10 transection SCI model. The spinal cord, including the lesion site, was removed at 14 days after SCI ( Fig. 2A). LINGO-1 expression was up-regulated significantly at 14 days after SCI in LINGO-1 +/+ mice, and there was no LINGO-1 expression at any time point in LINGO-1 −/− mice (Fig. S5A, B). Widespread apoptosis, as evidenced by increased expression of cleaved-caspase 3 protein in the cord tissue, was obvious, and was significantly improved at 14 days compared with 3 days after SCI (Fig. S5A, C). However, when LINGO-1 was deleted, the expression of cleaved caspase-3, which can induce apoptosis, was significantly down-regulated in the injured tissue of LINGO-1 −/− mice compared with LINGO-1 +/+ mice at each time point (Fig. 2B,C, Fig. S5A, C). TUNEL immunoreactivity was quantified at the following three regions of the spinal cord: rostral area, injury site, and caudal area. The percentage of TUNEL-positive cells in each area was calculated (Fig. S1). The results showed that the TUNEL-positive areas were significantly smaller in the LINGO-1 −/− group compared with those in the LINGO-1 +/+ group (Fig. 2D-F). This indicated that deletion of LINGO-1 can protect cells from apoptosis after SCI.

Rescue of oligodendrocytes and neurons in LINGO-1 KO mice
To determine whether apoptotic cells were associated with LINGO-1 expression, we co-labeled apoptotic cells around the injury site with LINGO-1 in LINGO-1 +/+ mice, which showed co-localization of LINGO-1 and TUNEL (Fig. 3E1-E4). Previous reports showed that LINGO-1 was present in oligodendrocytes and neurons (Mi et al., 2004;Mi et al., 2005). To determine the types of apoptotic cells, oligodendrocytes (APC) and neurons (MAP2) were each co-labeled with TUNEL. The results showed that the apoptotic cells were co-localized with APC ( Fig. 3A-B) and MAP2 (Fig. 3C-D). The sum of cells positive for APC/ TUNEL and MAP2/TUNEL was determined for the rostral area, injury site, and caudal area (Fig. S1). Compared with the LINGO-1 +/+ group, apoptosis in oligodendrocytes and neurons was significantly reduced in the LINGO-1 −/− group (Fig. 3F). These results suggested that LINGO-1positive cells underwent apoptosis in WT mice and that apoptosis occurred in oligodendrocytes and neurons, which was rescued by LINGO-1 KO.

Inflammation in the SCI site in LINGO-1 KO mice
To observe the inflammatory response following spinal cord transection, we evaluated IBA1 expression in WT and LINGO-1 KO mice at 14 days after SCI by immunofluorescence staining (Fig. 4A-B). IBA1 is a macrophage/microglia-specific calcium-binding protein that plays a key role in membrane ruffling and phagocytosis (Ohsawa et al., 2000). We found that IBA1-positive cells accumulated mainly at the SCI site  Huang, et al. Experimental Neurology 320 (2019) 112965 ( Fig. 4A2, B2). In addition, IBA1-positive cells were also detected in the rostral and caudal areas near the injury site (Fig. 4A1, A3, B1, B3) in the LINGO-1 +/+ and LINGO-1 −/− groups. IBA1 immunoreactivity was quantified in three regions: the rostral area, injury site, and caudal area (Fig. S1). There were no statistically significant differences in the number of IBA1-positive cells in the rostral and caudal areas of spinal cord between the LINGO-1 +/+ and LINGO-1 −/− groups. However, in the injury site, the number of IBA1-positive cells was significantly lower in the LINGO-1 −/− group (14.25 ± 5.17) compared with the LINGO-1 +/+ group (33.05 ± 3.46; Fig. 4A-C). Western blot was performed to quantify the level of inflammation in the injured spinal cord further. IBA1 expression was lower in the LINGO-1 −/− group (0.10 ± 0.04) than in the LINGO-1 +/+ group (0.43 ± 0.11; Fig. 4D, E). These results suggested that deletion of LINGO-1 can reduce the number of microglial cells/macrophages in the SCI site, resulting in reduction of inflammation after SCI.

Nerve fiber regeneration in the SCI site in LINGO-1 KO mice
At 14 days after SCI, immunofluorescence staining was performed to detect the expression level of NF, a nerve fiber marker, in the areas rostral and caudal to the injury site of the transected spinal cord. The results showed an increased amount of NF in the LINGO-1 −/− group compared to that in the LINGO-1 +/+ group (Fig. 6A-B). At higher magnification, immunofluorescence was noticeably more intense in the areas rostral and caudal to the SCI site in the LINGO-1 −/− group than that in the LINGO-1 +/+ group. Of note, the increase in NF-positive area in the injury site was more pronounced in the LINGO-1 −/− group (Fig. 6B2). NF-positive nerve fibers were parallel in the LINGO-1 +/+ group (Fig. 6A1, A3), while those in the LINGO-1 −/− group tended to be twisted in the rostral and caudal areas (Fig. 6B1, B3). In addition, quantitative evaluation (Fig. 6C) showed that the percentage of NFpositive areas in the rostral (54.02 ± 6.50), caudal (33.62 ± 5.02), and injury sites (23.12 ± 2.63) was significantly increased in the LINGO-1 −/− group when compared with the corresponding area in rostral (7.62 ± 1.74), caudal (2.80 ± 0.54), and injury sites (0.34 ± 0.21) in the LINGO-1 +/+ group. The results were consistent with Western blot results (Fig. 6D, E), which showed that NF expression at the SCI site in the LINGO-1 −/− group (0.54 ± 0.18) was significantly increased compared to that in the LINGO-1 +/+ group (0.20 ± 0.73). Many CGRP-positive ascending nerve fibers were also observed in the SCI site in LINGO-1 −/− mice, but 5-HT-positive descending nerve fibers appeared in large quantities at the boundary between rostral and injury site and only a small amount in the lesion site (Fig. 6G). In the injury site and boundary site of LINGO-1 +/+ mice, both CGRP-and 5-HT-positive nerve fibers were sparse (Fig. 6F). These data suggested that deletion of LINGO-1 promotes the regeneration of nerve fibers in the injured spinal cord, expecially CGRP positive nerve fibers.

Discussion
In the present study we investigated post-SCI cell survival, inflammation, glial scar formation, and nerve fiber regeneration in mice lacking LINGO-1. We demonstrated that LINGO-1 KO may decrease cell apoptosis, attenuate inflammation, and reduce glial scar formation. Furthermore, we provide evidence that LINGO-1 KO can enhance nerve fiber regeneration in the injury site after SCI.
In traditional genetically modified mice, modified exogenous DNA is inserted through homologous recombination in mouse embryonic stem cells (ESCs) using conventional gene-targeting methods, and the targeted ESCs are injected into WT blastocysts to generate chimeric mice containing the targeted gene modification (Capecchi, 2005). This method is costly and time-consuming . Co-injection of Cas9 mRNA and sgRNAs into zygotes also generates mice with biallelic mutations of targeted genes with high efficiency . Compared with previous gene-editing techniques, the CRISPR/ Cas9 system allows for rapid, efficient, and cost-effective genetic modification. Hence, this system has been widely used as a powerful genome-editing tool for producing genetically modified cell lines and animal models Platt et al., 2014;Wang et al., 2013). The CRISPR/Cas9 technology was suitable for generation of Previous studies showed that SCI induced cell death, inflammation, and glial scar formation, which inhibited nerve fiber growth and formation of synapses (Silver and Miller, 2004;Tran et al., 2018). SCI disrupts motor, sensory, and autonomic functions, which can cause limb paralysis (Lai et al., 2013;Lu et al., 2018;Ren et al., 2018). Michael et al. reported that cell apoptosis was observed 6 h after SCI, and lasted for many weeks, particularly oligodendrocyte apoptosis, which resulted in demyelination of white matter tracts (Beattie et al., 2000;Mizuno et al., 1998). Apoptosis occurs in two stages: the initial phase involves apoptosis of multiple cell types at the lesion center, and the later phase involves apoptosis of oligodendrocytes and microglial cells (Beattie et al., 2000). Apoptotic cells included neurons, oligodendrocytes, and microglial cells, but not astrocytes (Beattie et al., 2000;Crowe et al., 1997;Li et al., 1996;Shuman et al., 1997). LINGO-1 is a negative regulator of neuron and oligodendrocyte survival, nerve fiber regeneration, myelination, and functional recovery (Andrews and Fernandez-Enright, 2015). To investigate the protective role of LINGO-1 KO on SCI, LINGO-1 −/− mice underwent spinal cord transection. In the present study, LINGO-1 expression significantly increased from 3 to 14 days after SCI in WT mice, which corresponded with increased expression of activated caspase-3. Interestingly, we found that LINGO-1 KO significantly reduced the incidence of apoptosis in the injured spinal cord compared to that in WT mice. In LINGO-1 +/+ mice, apoptotic cells, including oligodendrocytes and neurons, expressed LINGO-1, and this apoptosis was rescued by deletion of LINGO-1. These results suggested that LINGO-1 contributed to cell apoptosis and that LINGO-1 KO may be a promising strategy to promote cell survival after SCI. These results were consistent with results presented by Ji et al. using a LINGO-1 antagonist (Ji et al., 2006). LINGO-1 KO may protect cells from apoptosis, by activating the EGFR/PI3-K/Akt pathway (Inoue et al., 2007), promotion of WNK3 kinase activity , upregulation of phospho-TrkB phosphorylation, and activation of the BDNF/TrkB signaling pathway (Fu et al., 2010;Fu et al., 2009) and decreasing caspase-3 expression and inhibition of RhoA/Rho-kinase (RhoA/Rock) pathway (Ji et al., 2006). In the present study, we demonstrated that LINGO-1 plays a direct role in cell apoptosis in injured spinal cord, but identification of surviving cells and signaling pathways involved in protection against apoptosis has not been performed. Increased cell survival may promote nerve fiber regeneration in LINGO-1 KO mice.
As documented previously by our group and others, SCI elicits acute inflammation, which is typically caused by physical injury and factors released from dead cells (Bianchi, 2007). In an acute inflammatory environment in the central nervous system, microglial cells migrate to the lesion area (Davalos et al., 2005;Gadani et al., 2015;Wu et al., 2005), resulting in a shift from M2-polarization to M1-polarization (Gwak and Hulsebosch, 2009;Kigerl et al., 2009). This polarization exacerbates inflammation, causing further loss of neurons and increased astrogliosis in the SCI site (Wang et al., 2015). IBA1 is a microglia−/macrophage-specific calcium-binding protein involved in cell membrane ruffling and phagocytosis (Ito et al., 1998;Ohsawa et al., 2000). Although inflammatory cells may contribute to clearing of cellular debris from the injury site and help to prevent the spread of the lesion (Hines et al., 2009), they also contribute to demyelination and are involved in nerve fiber injury (Weiner, 2008). Furthermore, cytokines released during the inflammatory response can damage oligodendrocytes and nerve fibers (Ruggieri et al., 2017). Therefore, novel strategies to decrease inflammation and promote effective recovery after SCI are of great interest. LINGO-1, a negative regulator, suppresses axonal regeneration, oligodendrocyte precursor cell maturation, and myelination in neurological disorders (Andrews and Fernandez-Enright, 2015;Foale et al., 2017). Our data showed that IBA1 expression in the SCI site after SCI in LINGO-1 −/− mice was markedly attenuated when compared to that in WT mice, indicating that absence of LINGO-1 in mice may negatively regulate the spread of inflammation in SCI.
Although downstream pathways of LINGO-1 were not examined in this study, a previous study by Paschalis Theotokis and colleagues showed that NgR, LINGO-1, and TROY complex were expressed in macrophages in the acute phase, and negatively regulated GAP-43 + axonal growth (Theotokis et al., 2016). These results agreed with our findings that LINGO-1 −/− contributed to inflammatory regulation by creating an anti-inflammatory microenvironment, which promoted nerve fiber regeneration.
Inflammatory cells infiltrating the SCI site activate resident glial cells, such as astrocytes and oligodendrocytes progenitor cells (Tran et al., 2018). Reactive astrocytes surrounding the lesion site create a wall-like structure with thick hypertrophied processes of overlapping outgrowths (Wanner et al., 2013), which forms the glial scar boundary (Orr and Gensel, 2018). The border formed by astrocytes also produces potently inhibitory molecules, such as CSPGs (Anderson et al., 2016;Orr and Gensel, 2018;Schachtrup et al., 2010). CSPGs are a family of extracellular matrix molecules up-regulated in the spinal cord after SCI (Jones et al., 2003;Shields et al., 2008). Up-regulation of CSPGs in central nervous system injury inhibits axonal regeneration (Hynds and Snow, 1999;Properzi et al., 2003;Silver and Miller, 2004). Thus, astrocytic glial scar forms a physical and chemical barrier to nerve fiber outgrowth (Huang et al., 2009;Yu et al., 2012). CSPGs consist of a core protein containing one or more unbranched polysaccharide glycosaminoglycan (GAG) chains which contribute to their inhibitory actions (Yu et al., 2012). Many studies have reported that glial scar can be inhibited by chondroitinase ABC (ChABC), an enzyme that selectively cleaves GAG chains from the protein core (Bradbury et al., 2002;Silver and Miller, 2004). Monnier et al. demonstrated that CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK signaling pathway (Monnier et al., 2003). Coles et al. reported that CSPGs inhibited nerve regeneration through inhibition of receptor protein tyrosine phosphatase sigma (RPTPσ) ectodomain oligomerization (Coles et al., 2011). In this study, we observed that deletion of LINGO-1 inhibited the formation of glial scar through down-regulation of GFAP and CSPGs after SCI, and promoted regeneration of nerve fibers in the SCI site, providing a source for ascending nerve fibers. The mechanism of reduction of glial scar in response to LINGO-1 deletion has not been characterized, but it may decrease GFAP expression by acting on its promoter through methylation and deacetylation . In addition, regeneration of nerve fibers containing NF and CGRP in the injury site may have originated from the surviving neurons after SCI. We suspect that more neurons had survived in the SCI, resulting in more regenerating nerve fibers in the lesion site and adjacent areas.

Conclusions
We observed that the LINGO-1 KO mice exhibited a lower incidence of cells undergoing apoptosis, decreased inflammation, and reduced glial scar formation, resulting in increased nerve fiber regeneration after SCI when compared with LINGO-1 +/+ mice. These results suggested that LINGO-1 deletion represents a novel approach to promote axonal regeneration. This may have been a result of reduction of cell apoptosis and improvement of the injury microenvironment through decreased inflammation and glial scar formation in the injured spinal cord. Therefore, inhibition of LINGO-1 may be a promising strategy for treatment of SCI to enhance cell survival and nerve regeneration.

Compliance with ethical standards
The use and care of animals were approved by the Ethics Committee of Sun Yat-sen University and carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institudes of Health.