Distinctive Alteration of Neuropeptide Y Expression Responsible for Neuro-Proliferation Following Zebrafish Spinal Cord Injury


 In strong contrast to the limited repair within the mammalian central nervous system, the spinal cord of adult zebrafish is capable of regeneration following injury. Understanding the mechanism underlying neural regeneration and functional recovery in spinal cord-injured zebrafish may lead to effective therapies for human spinal cord injury (SCI). Since neuropeptide Y (NPY) plays a protective role in the pathogenesis of several neurological diseases, in the present study, the effects of NPY on neuronal repair and subsequent recovery of motor function in adult zebrafish post-SCI were evaluated. Real-time quantitative PCR (qRT-PCR), in situ hybridization (ISH) and immunostaining of NPY revealed decreased NPY expression at 12 hours (h), 6 days (d) and 21 d after SCI. Double-immunostaining for NPY and Islet-1, a motoneuron marker, showed that NPY was expressed in spinal cord motoneurons. NPY morpholino (MO) treatment resulted in suppressed locomotor recovery and axon regrowth. PCNA and Islet-1 double-staining showed suppressed motoneuron proliferation in NPY-MO zebrafish. Similar to NYP, the mRNA level for NPY1R was also expressed within motoneurons and downregulated at 12 h and 21 d after SCI. Collectively, these data suggest that NPY expression in motoneurons promotes locomotor recovery and axon regrowth in adult zebrafish, possibly by regulating motoneuron proliferation through the activation of NPY1R.


Introduction
In mammals, spinal cord injury (SCI) is characterized by a lack of neuron regeneration throughout the lesion site, and results in permanent functional impairment caudal to the affected region. (Bareyre 2008;Cao and Dong 2013) However, different from mammals, motor neurons in adult SCI zebra sh demonstrated BrdU positive (Reimer et al. 2008), and adult zebra sh have a prominent regenerative capacity that enables the spinal cord to regenerate damaged nerves and recover locomotor function in a remarkably short period following SCI (Peng et al. 2017b;Liu et al. 2016;Fang et al. 2014). Since several molecular pathways are shared between zebra sh and mammals, zebra sh are considered to be an ideal SCI regeneration model for discovering key neural regeneration molecules for human SCI therapy .
Neuropeptide Y (NPY) is a 36-amino acid peptide that is highly conserved in vertebrates and widely expressed in both the central nervous system (CNS) and peripheral nervous system (Blomqvist et al. 1992;Gray et al. 1986;Tatemoto et al. 1982). NPY plays roles in several processes, including stress, food intake, anxiety, circadian rhythm, epilepsy, and chronic pain (Silva et al. 2002;Yoon and Kim 2019).
Importantly, NPY has been implicated in neuroprotection and neurogenesis (Malva et al. 2012;Duarte-Neves et al. 2016;Decressac and Barker 2012;Peng et al. 2017a;Chen et al. 2018). NPY plays a neuroprotective role in a mouse model of Alzheimer's disease, by inhibiting endoplasmic reticulum stressinduced cell death or intracellular oxidative stress through inhibiting caspase-3 and caspase-4 activities, and by increasing the level of BDNF and NGF . NPY is also able to exert neuroprotection in Parkinson's disease by inhibiting excitotoxicity (Decressac and Barker 2012), as well as regulate the survival and proliferation of different types of stem cells, including neural stem or precursor cells (Peng et al. 2017a).
In mammalian neural injury models, such as peripheral nerve injury or SCI, upregulation of NPY expression, either at the RNA or protein level, could be observed in the spinal cord (Fodor and Palkovits 1991;Zhang et al. 1993;Song et al. 2001;Coronel et al. 2017;Hwang et al. 2019). All of these studies have focused on NPY-related algesthesia research, because the dorsal horn of the spinal cord is functionally responsible for receiving sensory information. However, the function of NPY in neuro-repair and its alteration in zebra sh SCI remains unclear.
NPY performs its wide range of functions mainly via Y1, Y2, Y4, Y5 and Y6 receptors (Silva et al. 2002;Yi et al. 2018). Among these receptors, Y1 (NPY1R) is the most prevalent NPY receptor in the CNS and shows the highest a nity for NPY (Alexander et al. 2013). NPY also exhibits neuro-proliferative proprieties mainly via activation of NPY1R (Yi et al. 2018;Peng et al. 2017a). In addition, the zebra sh NPY1R corresponds structurally to its mammalian counterpart Salaneck et al. 2008;Fallmar et al. 2011;Matsuda et al. 2012), which suggests that NPY might function in mammals and zebra sh in a similar manner. Accordingly, these studies have led us to investigate the neuroprotective effect of NPY and NPY1R in the context of SCI in the zebra sh.

Animals
All experiments were approved by the Animal Care and Use Committees of Jiangnan University in China and carried out in accordance with the Guide for the Care and Use of Laboratory Animals (the eighth edition) from the National Institutes of Health. Wild-type adult zebra sh (Danio rerio, 6 months old) were bought from the Jiayu Aquatic Animals Company (Shanghai, China). All zebra sh (both sexes) used in the experiments were maintained on a 14 h light and 10 h dark cycle at 28°C, and were fed twice daily.

SCI surgery of adult zebra sh
Spinal cords of zebra sh were lesioned as described previously (SCI group) (Liu et al. 2016;Peng et al. 2017b;Fang et al. 2012). Brie y, sh were anesthetized in 0.033% tricaine methane sulfonate (MS-222, Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS), pH 7.4, until respiratory movement of the opercula stopped (around 3-5 mins). At the center between the dorsal n and the operculum, corresponding to the eighth vertebra (4-5 mm caudal to the operculum of the spinal cord), a longitudinal incision was made through the muscle layer, and the vertebral column was exposed. The spinal cord was cut completely between the two vertebrae with micro-scissors, then the wound was sealed with Histoacryl (B. Braun, Melsungen, Germany). Then, sh were kept individually in 28°C water, Fish resumed breathing within a few seconds. The sham-injured control (Sham) underwent the identical surgical procedure except that the spinal cord was not severed. All surgical procedures were performed on ice under a microscope.
The schematic diagram for SCI and tissue preparation for other experiments is shown in Supplemental

qRT-PCR for NPY and NPY1R
Total RNA was extracted from the 4 mm segment of spinal cord, caudally extending from the lesion site, with an EZgene TM Tissue RNA Miniprep Kit (Biomiga, San Diego, CA, USA) according to the manufacturer's instructions. First-strand cDNA was generated using random primers and a PrimeScript TM RT Master Mix (Perfect Real Time) (TAKARA BIO, Otsu, Japan). qRT-PCR was carried out with SYBR® Premix Ex Taq TM II (Tli RNase H Plus) (TAKARA BIO, Otsu, Japan), and the comparative cycle threshold Ct method (∆∆Ct method) was applied for data analysis. Primers for qRT-PCR were designed using Primer Express 5.0 software (Applied Biosystems, Foster City, CA, USA). All experiments were performed in duplicate, and PCR products were validated by melting curves to con rm the presence of single PCR products. GAPDH served as the internal control. Primers used were as follows: zebra sh NPY forward: GACTCTCACAGAAGGGTATCC, reverse: GGTTGATGTAGTGTCTTAGTGCTG. NPY1R forward: GTAGCAGTGAGTCTAAACGGATAA, reverse: GAAGTTGCGGTTCAGAAAGC. GAPDH forward: GTGTAGGCGTGGACTGTGGT, reverse: TGGGAGTCAACCAGGACAAATA. (Pei et al. 2007) A no-template control group was performed in each experiment using the same reaction conditions at different time points. The number of sh for this assay was 10 for each group.

ISH for NPY and NPY1R
In situ hybridization (ISH) probes (sense and anti-sense) for NPY and NPY1R mRNA were transcribed in vitro. Puri ed PCR fragments were cloned into the pGM-T vector (Tiangen, Beijing, China), and the sequences were veri ed by sequencing. Digoxigenin (DIG)-labeled sense and anti-sense RNA probes were generated using the MEGAscript system (Ambion, Austin, TX, USA) according to the manufacturer's instructions. Non-radioactive detection of mRNA in sections of the adult zebra sh was performed as described with slight modi cations (Liu et al. 2016;Fang et al. 2014). At different time after SCI, wild-type zebra sh were deeply anaesthetized with 0.033% MS-222, then a 4 mm spinal cord segment, extending caudally from the lesion, was rapidly removed and xed with 4% paraformaldehyde in PBS at 4°C for 12 h, then incubated in 15% sucrose in PBS overnight at 4°C. The spinal cord tissue was prepared as frozen sections at 10 mm thickness longitudinally, and the sections through the central canal were used for ISH. DIG-labeled sense and anti-sense RNA probes for NPY (NM_131074.2) and the NPY1R (NM_001102391.1) were generated by in vitro transcription using T7 and SP6 RNA polymerases. The frozen sections were initially pre-hybridized for 2 h at 55°C, and then hybridized with the DIG-labeled probes for 18 h at 55°C. After hybridization, the sections were washed and blocked, then alkaline phosphatase-coupled anti-DIG fragment antibodies (Roche, Indianapolis, IN, USA) were applied for 1 h at room temperature and developed with NBT/BCIP (nitro-blue-tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) (Roche, Indianapolis, IN, USA). Following staining, sections were imaged using a microscope (Eclipse 80i, Nikon, Tokyo, Japan). Five sh were analyzed for each group.

Inhibition of NPY expression by morpholino treatment
Morpholinos (MOs) have high mRNA binding a nity and exquisite speci city, and can modify pre-mRNA splicing in the nucleus by targeting splice junctions or splice regulatory sites. In this study, a spliceblocking MO was used to knock down NPY expression. The NPY anti-sense MO (NPY MO group) (5'-TACAAACGAGGAGCTCACCTCTGCC-3', Vivo-Porter-coupled) and standard control MO (CT MO group) (5'-CCTCTTACCTCAGTTACAATTTATA-3', Vivo-Porter-coupled) (Gene Tools, Philomath, OR, USA) were dissolved in Danieau solution and absorbed by small pieces of gelfoam (Upjohn, Kalamazoo, MI, USA).
The gelfoam was allowed to dry and then was divided into smaller pieces, each yielding 800 ng of MO, which were then inserted into the transection site of the spinal cord immediately after SCI. Each animal was allowed to survive the surgery for 6 weeks.

Swim-tracking of zebra sh
A swim-tracking test was used to assess the locomotor recovery of zebra sh at 1, 2, 3, 4 and 6 weeks after SCI, as described previously (Fang et al. 2014). In each trial, a zebra sh was placed in a brightly illuminated tank (22.5×11×11 cm) lled with aquarium water (5 cm deep) at 25°C. The total swimming distance of the zebra sh within a 5-min period was recorded with video tracking and EthoVision XT software (Noldus, Wageningen, Netherlands). The number of sh analyzed for this assay was twelve for each group.
Anterograde tracing of zebra sh motoneurons at 6 weeks following MO treatment To study whether regenerated axons had crossed the lesion site, biocytin (Sigma, St. Louis, MO, USA) was applied via a second surgery at the site of the brain stem/spinal cord junction at 6 weeks after SCI (Becker et al. 1997) (Fig 3. A). Biocytin was dissolved to approximately 1 mg/10 µl PBS. A small piece of gelfoam saturated with either vehicle or biocytin was put into the secondary injury site. Then the injury site was seal by Histoacryl (B. Braun, Melsungen, Germany). After 24 h, 4 mm of the spinal cord segment just beneath the rst injury site was collected (Fig 3. A) and xed in 4 % paraformaldehyde in PBS at 4°C overnight, embedded in 15 % sucrose, and sectioned coronally (in 25 μm-thick sections) or longitudinally (in 16 μm-thick sections) on a cryostat. Biocytin labeling was detected with streptavidin-Cy3 (1:400, Bioss, Beijing, China) and imaged with a uorescence microscope. The intensity of biocytin-positive axons re ects the axonal regrowth. Five random coronal sections through the central canal in each sh were measured to obtain mean density values for immuno uorescence with Image J software (Wayne Rasband, National Institutes of Health, USA). Five sh were in each group (sham, control MO, NPY MO group) (Fang et al. 2012).
Co-localization of NPY1R mRNA with NPY or motoneurons by immuno uorescence and ISH To co-localize NPY1R with NPY and motor neurons, cells expressing NPY1R mRNA were identi ed by ISH (we were unable to obtain antibodies capable of speci cally recognizing zebra sh NPY1R, thus immunohistochemistry was not performed), and NPY and motoneurons were identi ed by immunohistochemistry. ISH was performed as described above. Following ISH, the sections were processed using standard immunohistochemical procedures. In brief, spinal cord sections prepared as above were incubated with primary antibody against rabbit NPY and mouse Islet-1 at 4°C overnight, followed by incubation with FITC-conjugated goat anti-rabbit/mouse secondary antibody at room temperature for 2 h. Fluorescence was observed with a uorescence microscope. Five sh were used for each group.
Statistical Analysis SPSS 19.0 software was used for data analysis (two-way ANOVA or one-way ANOVA). All data are presented as means ± SEM. The level of signi cance was set at p < 0.05 for the threshold for signi cance (*p < 0.05, **p < 0.01, ***p < 0.001). All experiments were performed in triplicate.

SCI induces decreased NPY expression in the adult zebra sh spinal cord
To examine the possible involvement of NPY in spinal cord regeneration of adult zebra sh, the expression of NPY in the 4 mm segment of spinal cord immediately caudal to the lesion site was detected by qRT-PCR at 12 h, 6 d, 21 d after SCI. We found that compared to the sham group, expression of NPY mRNA in the SCI group was signi cantly decreased to 26.9% at 12 h (***p < 0.001), 80.3% at 6 d (*p < 0.05) and 42.1% at 21 d (**p < 0.01), with NYP expression 6 d post-SCI showing a transient peak (Fig. 1A). The qRT-PCR results were con rmed by ISH where, compared to the sham group, NPY mRNApositive signals in SCI group were less at 12 h, 6 d and 21 d, especially in the large cells along the central canal, which were inferred to be motoneurons based on the morphology (Fig. 1B). Immuno uorescence analysis also showed that the NPY protein level in the SCI group was less than the sham control at 12 h, 6 d and 21 d. NPY-positive cells were located predominantly in the area surrounding the central canal ( Fig. 1C).

NPY knock-down retards locomotor recovery following SCI in zebra sh
To determine the locomotor recovery of NPY MO-treated adult zebra sh (NPY MO group) following SCI, we measured weekly the distance that the sh could swim in a period of 5 min. Standard control MO was used as the control (CT MO group). By qRT-PCR analysis, NPY mRNA expression was found to be reduced at 3 d and persisted for 1 w after SCI in the NPY MO group ( Fig. 2A), con rming knock-down of NPY. At 1 w after SCI, no difference in locomotion was observed between the NPY MO and control MO groups. However, NPY MO zebra sh swam a shorter distance in 5 min than CT MO zebra sh at 2, 3 and 6 w ( Fig  2B) after SCI. At 2 w after SCI, the total distance moved by the NPY MO group (200.73 ± 17.36 cm) was 68% of that in the CT MO group (295.34 ± 35.84 cm, *p < 0.05). At 3 w, the total swimming distance of the NPY MO group (216.96 ± 35.12 cm) was 57.8% of that in CT MO group (375.08 ± 38.08 cm, ***p < 0.001), and at 4 w after SCI, the total distance moved by the NPY MO group (272.69 ± 67.11 cm) was 48.5 % of that in the CT MO group (561.86 ± 91.96 cm, *p < 0.05). Similarly, at 6 w after SCI, the total distance moved by the NPY MO group (267.33 ± 86.62 cm) was further reduced to 36.6 % of the control group (730.07 ± 119.42 cm, *p < 0.05) (Fig. 2C). These results indicate that the recovery of locomotor function of SCI zebra sh is inhibited by NPY MO treatment, suggesting that NPY may promote locomotor recovery.

Inhibition of NPY impairs axonal regrowth after SCI in zebra sh
To further elucidate the requirement of zebra sh for NPY in recovery from SCI, we examined axonal regrowth beyond the lesion site by anterograde tracing at 6 w post-SCI (Fig. 3A). An uninjured group was used as the normal control. Biocytin-positive signals in both coronal and sagittal sections from the NPY MO group were considerably less than that of uninjured-and the CT MO-group (Fig. 3B). In coronal sections, compared with uninjured group, the CT MO and NPY MO groups showed an 8.5% (**p < 0.01) and 32.2% (***p < 0.001) decrease of immuno uorescence intensity, respectively. Compared with the CT MO group, the NPY MO group showed a 25.3% decrease in axonal regrowth (***p < 0.001) (Fig. 3C). These observations support the view that NPY promotes axonal regrowth, and enhances locomotor recovery during regeneration after SCI in zebra sh.

NPY MO treatment reduces proliferation of motor neurons in the spinal cord following SCI
To characterize the predominant NPY-positive cell types, we performed double-immuno uorescence labeling for NPY and Islet-1 (a marker for motoneurons) at 12 h and 21 d after SCI. The results showed that NPY was expressed in motor neurons. Moreover, two subtypes of motoneurons, of small and large size, were observed (Fig. 4A). To reveal the effect of NPY on motoneurons, double immunostaining of PCNA and Islet-1 was used after NPY MO treatment. We found that CT MO-treated zebra sh showed more proliferating motor neurons than the sham group (Fig. 4B), indicating that neurogenesis occurred following SCI in zebra sh. NPY MO-treated zebra sh showed reduced numbers of proliferating motor neurons than CT MO-treated zebra sh (Fig. 4B), indicating the reduced neurogenesis was induced by NPY knock-down. These ndings prompted us to speculate that NPY could work as a modulator of motoneuron proliferation.

SCI induces decreased NPY1R expression in the zebra sh spinal cord
Given that NPY possesses the capacity to modulate neurogenesis and has previously been implicated in proliferation through activating the NPY1R subtype (Hansel et al. 2001), we sought to determine whether NPY could exert its effects on motor neuron regeneration through NPY1R. ISH and qRT-PCR was used to detect the expression of NPY1R in the 4 mm segment of spinal cord including and caudal to the lesion site at 12 h, 6 d, 21 d after complete spinal cord lesion. We found the expression of NPY1R mRNA in the spinal cord of the SCI group decreased to 47.4% at 12 h (*p < 0.05), 37.2% at 6 d (***p < 0.001) and 51.6% at 21 d (*p < 0.05) compared to the sham control (Fig. 5A). The qRT-PCR results were con rmed by ISH. Compared to the sham control, NPY1R mRNA expression was lower at 12 h, 6 d, and 21 d after SCI (Fig.   5B). The change in expression of NPY1R was consistent with that of NPY.

NPY1R co-localizes with NPY and motor neurons
Co-localization of NPY1R mRNA (due to the absence of zebra sh NPYR1 antibody for zebra sh) and NPY protein were determined by ISH and immuno uorescence, respectively, showed that NPY1R co-localizes with most NPY-positive cells at 12 h and 21 d after SCI. SCI zebra sh showed fewer co-labeled cells than the sham group (Fig. 6A). In addition, ISH for NPY1R mRNA and Islet-1 immunostaining for motoneurons, in the same longitudinal sections of the spinal cord, displayed co-localization at 12 h and 21 d after SCI. Cells showing co-localization were fewer in SCI zebra sh than in sham-treated zebra sh (Fig. 6B). NPY and NPY1R showed the same expression trend. These results indicate that NPY1R mRNA is expressed in spinal cord motoneurons, suggesting that NPY is likely to exert its effects on motoneuron proliferation through the NPY1R.

Discussion
Molecules that promote regeneration and functional recovery following zebra sh SCI could provide effective therapeutic clues for human SCI. NPY is widely distributed throughout all levels of the spinal cord in a variety of mammals, including the rat, guinea pig, cat, marmoset, and horse (Gibson et al. 1984), and also functions as a neuroprotective peptide against excitotoxicity and apoptosis (Decressac and Barker 2012;Malva et al. 2012;Duarte-Neves et al. 2016). In the zebra sh CNS, NPY has been reported to be expressed in the hypothalamus and medulla (Mathieu et al. 2002;Jeong et al. 2018), but its expression in zebra sh spinal cord has not been described. Here, we explore the changes of NPY and the NPY1R, and their function in zebra sh SCI neuro-repair.
We found that in both normal control zebra sh and SCI zebra sh, NPY and NPY1R are present in spinal cord motoneurons. Morphologically, in normal control zebra sh, both small and large size motoneurons were present, with the large motoneurons being in the majority. Following SCI, in the early post-SCI stage (12 h post-SCI), NPY-positive motoneurons mainly displayed a small-sized appearance along the central canal, whereas in the late post-SCI stage (21 d post-SCI), NPY-positive motoneurons were mainly largesized, which is in line with that in rats following axotomy (Zhang et al. 1993). Both large and small motoneurons were NPY-positive. The two types of motoneurons might correspond to early-developing primary (small-size, immature) and late-developing secondary (large-size, mature) motoneurons after spinal lesion (Grunwald et al. 1988) and persist through adulthood in the zebra sh (Stil and Drapeau 2016). It has been reported that NPY is expressed in both immature and mature neurons, and might function in modulation of proliferation in immature neurons and neuroprotection in mature neurons (Decressac and Barker 2012). Numerically, both NPY and NPY1R are down-regulated at the injury site following SCI. NPY shows down-regulation in spinal cord motoneurons at 12 h, 6 d and 21 d post-SCI, which is contrary to ndings in mammalian neural injury. In patients or mammalian models suffering from peripheral nerve injury (such as transection, loose ligation, a crushed sciatic nerve or its branches, or chronic constriction injury of the infraorbital nerve), upregulation of NPY in the dorsal root ganglia (DRG) and spinal cord dorsal horn (SCDH, downstream of the DRG in the nociceptive afferent pathway) following injury has been reported (Brumovsky et al. 2007;Diaz-delCastillo et al. 2018). Since NPY is closely related with nociceptive afferent neurons, the underlying mechanism might be due to peripheral nerve injury causing NPY up-regulation in DRG neurons (hub of the nociceptive afferent pathway), which further leads to increased NPY transport from the DRG to the SCDH, manifested as the upregulation of NPY in the SCDH (Brumovsky et al. 2007;Diaz-delCastillo et al. 2018). In mammalian SCI models, several studies have described the upregulation of NPY, NPY1R and NPY2R in the spinal cord (Song et al. 2001) (Coronel et al. 2017;Hwang et al. 2019). Hwang and colleagues described the upregulation of NPY in the rat DRG following SCI. SCI rats, that had received transplantation of GDNF-hNSPCs (human neural stem progenitor cells overexpressing glial cell line-derived neurotrophic factor), show a reduction of glial scar formation, and enhanced neurite outgrowth and axonal extension. Interestingly, the upregulation of NPY in the DRG is also reversed (Hwang et al. 2019). All previous studies of NPY, NPY1R and NPY2R in mammalian neural injury models have only focused on their alteration and roles in nociceptive afference, but their function in neuro-repair was not involved (Song et al. 2001;Coronel et al. 2017;Hwang et al. 2019). Using a zebra sh SCI model, we explore the function of NPY in neuro-repair. When NPY is knocked-down by MO treatment, locomotor recovery, axon regrowth and neuro-proliferation are inhibited. Hence, our observations suggest that NPY is a bene cial regulator in the progression of zebra sh spinal cord regeneration, probably by promoting neuro-proliferation via NPY1R. The contrary expression of NPY and NPY1R at the injury site in mammalian and zebra sh neural injury models suggests a special role for NPY in zebra sh SCI recovery. A comparative study concerning different gene regulation mechanisms between species should also be explored.
NPY has been reported to stimulate neural cell proliferation, as well as enhance growth and survival (Decressac et al. 2009;Palkovits 1995). It exhibits neuro-proliferative properties and regulates in ammation mainly by activating the NPY1R (Hansel et al. 2001;Howell et al. 2005;Milenkovic et al. 2004;Decressac et al. 2009;Peng et al. 2017a;Yi et al. 2018;Wheway et al. 2007;Malva et al. 2012;Farzi et al. 2015). In our study, NPY1R expression was also detected. Consistent with NPY expression, NPY1R is also downregulated at 12 h, 6 d, and 21 d following SCI. By immunostaining, we showed that NPY1R staining overlaps with most NPY-positive motoneurons in the spinal cord. Thus, our data favor the possibility that NPY may promote motoneuron proliferation mainly by activating NPY1R in zebra sh.
As a widely expressed peptide in both the CNS and PNS, NPY was also found to regulate the release and function of dopamine and serotonin (Myers et al. 1992;Quarta and Smolders 2014), which are also reported to involved in neural repair (Barreiro-Iglesias et al. 2015;Chapela et al. 2019;Cigliola et al. 2020). The further study on the mechanism and application of NPY in neural repair is needed.
In conclusion, our results suggest that NPY may play a bene cial role in neuro-recovery from SCI by promoting axonal regrowth and regulating motoneuron proliferation via the NPY1R in adult zebra sh. The different expression patterns of NPY and NPY1R between mammalian and zebra sh SCI models might contribute to the different capacities for neuro-regeneration. The profound mechanism and detection of NPY and NPY receptors over a longer period needs to be further explored. In addition, NPY is of particular interest as it has the ability to cross the blood-brain barrier (Kastin and Akerstrom 1999), in contrast to most of the neurotrophic and neuro-proliferative factors identi ed so far. Thus, adjunctive therapies with appropriate molecules, such as NPY, NPY1R agonists or pharmacological derivates, may be of signi cant potential to manage SCI therapy.     control MO and NPY MO groups. The total swimming distance of zebra sh in 5 mins was reduced to 70.4%, 42.3%, 48.9% and 42.3% at 2 w, 3 w, 4 w and 6 w respectively, after SCI in the NPY MO groups vs.
Values represent means ± SEM.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.