Peptide OM-LV20 promotes structural and functional recovery of spinal cord injury in rats

At present, there are no satisfactory therapeutic drugs for the functional recovery of spinal cord injury (SCI). We previously identi�ed a novel peptide (OM-LV20) that accelerated the regeneration of injured skin tissues of mice and exerts neuroprotective effects against cerebral ischemia/reperfusion injury in rats. Here, the intraperitoneal injection of OM-LV20 (1 µg/kg) markedly improved motor function recovery in the hind limbs of rats with traumatic SCI, and further enhanced spinal cord repair. Administration of OM-LV20 increased the number of surviving neuron bodies, as well as the expression levels of brain-derived neurotrophic factor and its receptor tyrosine receptor kinase B. In the acute stage of SCI, OM-LV20 treatment also increased superoxide dismutase and glutathione content but decreased the levels of malonaldehyde and nitric oxide. Thus, OM-LV20 signi�cantly promoted structural and functional recovery of SCI in adult rats by increasing neuronal survival and BDNF and TrkB expression, and thereby regulating the balance of oxidative stress. Based on our knowledge, this research is the �rst report on the effects of amphibian-derived peptide on the recovery of SCI and our results highlight the potential of peptide OM-LV20 administration in the acceleration of the recovery of SCI.


Introduction
Traumatic spinal cord injury (SCI) often results in severe nerve defects and dysfunction due to secondary injury and limited self-repair ability of the central nervous system.Patients with SCI often have different degrees of motor, sensory, and autonomic dysfunction.In severe cases, respiratory, urinary, and other systems may be affected, which can be life-threatening.These factors not only result in serious physical and psychological harm but can also cause huge economic burden to the patients, their families, and society as a whole 1 .The total global incidence of traumatic SCI is 10.5 cases per 100 000 people, reaching as high as 13.69 cases per 100 000 people in high-income countries, and the incidence rate continues to increase year by year 2,3 .
After SCI, a series of primary and secondary pathological changes, such as ischemia and anoxia, electrolyte changes, free radical production, and lipid peroxidation, lead to neuronal apoptosis and necrosis, axon dissolution, and excessive microglia and astrocyte activation 4 .In the acute phase of injury, high in ammatory cell in ltration leads to a continuous in ammatory response and increased oxidative stress, which aggravate tissue damage and further deteriorate the structure and function of the spinal cord after injury 5 .Therefore, it is important to reduce secondary injury and improve the local microenvironment to promote function and circuit reconstruction after SCI 6 .At present, however, there is no speci c drug treatment to solve these issues.The steroid drug methylprednisolone (MPED), which is widely used in the treatment of SCI, exhibits a better anti-in ammatory effect when used in large doses in the early stage of injury, but its side effects and therapeutic results have been questioned 7 .Therefore, the development of new drugs to promote SCI repair and recovery is critical.
Many peptides demonstrate high activity, high speci city, safety, low cost, and easy production 8 .A growing number of pharmacologically active peptides have also been identi ed, including antimicrobial 9 , antioxidant 10 , analgesic 11 , wound-healing 12 , and anti-gout peptides 13 .We previously identi ed a peptide OM-LV20 (amino acid sequence: LVGKLLKGAVGDVCGLLPIC) from amphibian skin secretions, and functional results indicated that OM-LV20 accelerated the recovery of skin and regulated the levels of blood glucose in mice 14,15 .At the same time, our previous research found that peptide OM-LV20 exerts neuroprotective effects on cerebral ischemia reperfusion injury in rats.meanwhile, indicated that OM-LV20 had high stability and could through the MCAO-damaged blood-brain barrier 16 .Thus, based on the activities and characteristics of OM-LV20 and the pathological changes experienced in SCI, we applied this peptide for SCI treatment in adult Sprague-Dawley (SD) rats.Results showed that the application of OM-LV20 improved the microenvironment of the injured spinal cord and promoted motor function recovery of the hind limbs.Therefore, this study con rmed that natural active peptides could be used in the treatment of traumatic SCI, thus providing a new template for the development of candidate drugs to promote SCI repair.

Synthesis of OM-LV20
OM-LV20 has an amino acid sequence of 'LVGKLLKGAVGDVCGLLPIC' and an intramolecular disul de bridge between 14th C and 20th C. OM-LV20 was arti cially synthesized using solid phase synthesis and commercially provided by Wuhan Bioyeargene Biotechnology Co. Ltd. (Wuhan, China).

Animals
The adult SD rats (female, 230-250 g, 8-9-weeks old) used in this study were provided by the Animal Experimental Center of Kunming Medical University.The rats were housed individually in cages with free access to water and food.The animals were acclimatized to the environment for 7 days prior to surgery.
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Kunming Medical University, China (SYXK (Yunnan) K2015-0002).
The surgical procedures have been described in detail in previous research 18 .In brief, the spinal cord was completely transected at thoracic level Th10.When the spinal cord was fully exposed, it was hooked with a blunt hook needle and cut with ophthalmic scissors.Transection was repeated three times on the cross-section with a scalpel to ensure that the spinal cord was completely transected.For the sham-operated group, only the vertebral arch plate was opened.After the operation, the wound was locally coated with penicillin sodium powder.After the rats awakened, they received a dose of penicillin sodium (40 U/kg, intramuscular (i.m.) injection) twice a day until there was no hematuria.

Postoperative care and treatment
After surgery, all rats were maintained in single cages in a SPF feeding room and were cared for by nursing staff.The bladder was emptied manually thrice a day until the voiding re ex was re-established.The MPED group received an i.p. injection once every 8 h after surgery (three times).The OM-LV20 and saline groups received i.p. administration once a day for 14 days after surgery.

Behavioural assessment
The Basso-Beattie-Bresnahan (BBB) scale was applied to evaluate the recovery of motor function of the hind limbs in rats.Brie y, three specially trained raters conducted double-blind evaluation on the day of surgery (after waking) and 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks after surgery.The rats were placed on an open scoring site and allowed to move freely for 5 min.The raters observed the rats and then evaluated their performance according to the BBB scale 19 .

Histological evaluation
Four weeks after surgery, rats were euthanized with sodium pentobarbital (200 mg/kg, i.p.) and perfused intracardially with 200 mL of 0.9% saline, followed by 250 mL of 4% paraformaldehyde.Taking the injured site as the center, 2 cm of intact spinal cord was left at the head and tail, respectively, and photos were taken.After xation and dehydration, frozen embedding methods were used to embed the spinal cord tissue and store in -80 ℃ refrigerator for future use.

Hematoxylin and eosin (H&E) staining
Spinal cord tissue was cut into 8-µm thick sections along the coronal plane, then stained with H&E.The staining results were observed and photographed using a microscope, and the tissue defect degree was analyzed by Image J 20 .

Nissl staining
Spinal cord tissue was cut into 8-µm thick sections along the horizontal plane of the caudal end of the glial scar and the coronal surface of the injury site.The sections were then attached to glass slides, followed by Nissl staining (Solarbio, G1430, China), absolute ethanol dehydration, xylene transparency, and neutral gum sealing.The staining results were observed and photographed under a microscope, and the survival of neuronal cells was analyzed using Image J 21 .2.9.Immunohistochemical staining Spinal cord tissue was cut into 8-µm thick sections along the coronal plane.The sections were attached to glass slides, Rinse in distilled water, soak in PBS for 5 minutes, incubate in 3% H2O2 deionized water for 15-20 minutes to eliminate endogenous peroxidase activity, rinse in PBS, 3 times, 3 minutes each time, add blocking solution (20% normal goat serum) ), incubate at room temperature for 15-20 minutes, pour off, drop primary antibodies, overnight at 4ºC, rinse with PBS, 3 times, 3 minutes each time, drop polymer adjuvant, incubate at 37°C for 15-20 minutes, rinse with PBS, 3 times, 3 minutes each time, drop horseradish enzyme labeled anti-rabbit IgG polymer, incubate at 37 ºC for 15-20 minutes, rinse with PBS, 3 times, 3 minutes each time, stained with DAB, washed with distilled water, counterstained with hematoxylin, dehydrated, transparently mounted, and sealed with neutral gum.The primary antibodies used for BDNF and TrkB were anti-BDNF rabbit pAb (1:150, A nity Biosciences, DF6387, China) and anti-TrkB rabbit pAb (1:200, Proteintech, BC031835, China), respectively.A rabbit SP kit (SP-0023, Bioss, China) was used for secondary antibody detection.The staining results were observed and photographed under a microscope, and analyzed with Image J 21 .

Enzyme-linked immunosorbent assay (ELISA)
Four weeks after surgery, rats were euthanized with sodium pentobarbital (200 mg/kg, i.p.) and perfused intracardially with 200 mL of 0.9% saline.The rats were then immediately placed on ice to remove the tissues of interest.With the injured site at the center, 1 cm of complete spinal cord was removed at the head and tail ends, respectively, then washed with precooled PBS (0.01 M, pH = 7.4) to remove residual blood clots, weighed, placed in sterile enzyme-free centrifuge tubes, and stored at − 80 ℃.The stored spinal cord tissues were then examined using a BDNF enzyme-linked immunosorbent kit (Jiang Lai Bio, JL12910, China).The Excel program was used to draw a standard regression curve and calculate the concentration of samples in different groups 21 .

Determination of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and nitric oxide (NOx) content
At 48 h after surgery, rats were anesthetized with pentobarbital sodium (200 mg/kg, i.p.), with 0.9% saline used for cardiac perfusion.The rats were then immediately placed on ice to collect the L-2 and S-1 spinal cord segment.The nerve root was cut and the spinal cord segment between L-2 and L-5 was preserved, with the tissue rinsed with prechilled PBS (0.01 M, pH = 7.4) to remove residual blood clots, then weighed, placed in sterile enzyme-free centrifuge tubes, and stored at − 80 ℃.The stored spinal cord tissues were then examined via relevant kits to determine MDA (Suzhou Grace Biotechnology Co. Ltd., G0109W, China), SOD (Suzhou Grace Biotechnology Co. Ltd., G0101W, China), GSH (Suzhou Grace Biotechnology Co. Ltd., G0206W, China), and NOx content (Suzhou Grace Biotechnology Co. Ltd., G0803W, China) 20 .

OM-LV20 signi cantly improved hind limb motor function in adult SD rats
During the 4-week observation period, the BBB scoring method was used to evaluate motor function recovery in SCI rats and number of deaths were also recoded.There were 6, 6, 10, 10, 10, 11, and 9 surviving rats in the normal, sham-operated, saline, MPED, OM-LV20 (10 ng/kg), OM-LV20 (100 ng/kg), and OM-LV20 (1 µg/kg) groups, respectively.The BBB scores are shown in Fig. 1A.From the operation to 4 weeks after, hind limb motor function in the SCI group recovered gradually, but not fully.In the rst week after SCI, hind limb motor function in the OM-LV20 and MPED groups improved signi cantly compared with that in the saline group; at 4 weeks, the scores were 12.15 ± 1.88 for the saline group (n = 60), 12.88 ± 0.85 for the MPED group (n = 60), and 15.55 ± 1.23, 14.91 ± 1.98, and 15.56 ± 1.70 for the OM-LV20 groups (10 ng/kg, n = 60; 100 ng/kg, n = 66; 1 µg/kg, n = 54, respectively).The survival rates are shown in Fig. 1B.From days 3 to 14, some injured rats died due to infection, bowel obstruction, and sores.From day 14, the rats in each group passed the dangerous period, and the death rate decreased signi cantly, although the MPED group exhibited a signi cantly higher death rate than the other groups.At day 28, the survival rate in the MPED group was only 40.00%, whereas that in the saline group was 62.50%, OM-LV20 (10 ng/kg) group was 66.67%, OM-LV20 (100 ng/kg) group was 68.75%, and OM-LV20 (1 µg/kg) group was 69.23%.The percentage change in postoperative body weight is shown in Fig. 1C.After the operation, the body weights in each group decreased, with the MPED group showing the greatest loss.
3.2.OM-LV20 signi cantly reduced secondary injury of spinal cord tissue Four weeks after surgery, the head and tail of the transected spinal cord grew together, and an obvious glial scar was observed (Fig. 2).However, the continuity of the two ends of the glial scar in the saline group was signi cantly worse than that in the OM-LV20 and MPED groups, and the glial scar in the saline group was signi cantly longer.As shown in Fig. 2A, obvious glial scars formed in each group and obvious defects remained in the tail of the glial scars in the saline group (shown by the blue arrow).Figure 2B shows the lengths of the local glial scars in each group, which were 8.50 ± 1.06 mm in the saline group (n = 4), 6.11 ± 1.14 mm in the MPED group (n = 7), 5.09 ± 0.73 mm in the OM-LV20 (10 ng/kg) group (n = 9), 5.26 ± 0.82 mm in the OM-LV20 (100 ng/kg) group (n = 5), and 5.38 ± 0.87 mm in the OM-LV20 (1 µg/kg) group (n = 10).We speculate that application of OM-LV20 might reduce secondary injury of spinal cord tissue in the acute phase and improve nerve cell survival at the injured site.This speculation was con rmed in the follow-up H&E staining and acute phase biochemical testing results.

OM-LV20 increased survival of neuronal cell bodies at injury site
Staining of the caudal area of the glial scars showed that there were fewer neuronal cell bodies in each group (Fig. 4); however, compared to the OM-LV20 and MPED groups, the number of neuronal cell bodies in the saline group decreased signi cantly (Fig. 4B, black arrow refers to neuronal cell bodies).Figure 4C shows the number of surviving neuronal cell bodies in each group, i.e., 6.22 ± 0.83 in the saline group (n = 9), 11.00 ± 1.12 in the MPED group (n = 9), 9.67 ± 1.23 in the OM-LV20 (10 ng/kg) group, 10.00 ± 1.41 in the OM-LV20 (100 ng/kg) group, and 11.33 ± 1.80 in the OM-LV20 (1 µg/kg) group.The coronal surface staining is shown in Fig. 5. Results indicated that there were fewer neuronal cell bodies in the injured area; however, compared to the OM-LV20 and MPED groups, the number of neuronal cell bodies in the saline group decreased signi cantly (Fig. 5B, black arrow refers to neuronal cell bodies).Figure 5C shows the number of surviving neuronal cell bodies in each group, i.e., 0.78 ± 0.67 in the saline group (n = 9), 2.44 ± 0.73 in the MPED group (n = 9), 1.00 ± 0.71 in the OM-LV20 (10 ng/kg) group, 1.56 ± 0.56 in the OM-LV20 (100 ng/kg) group, and 2.67 ± 0.71 in the OM-LV20 (1 µg/kg) group.These results indicate that neurons showed good survival after treatment with the active peptide (OM-LV20) and MPED.Thus, survival status and number of neuronal cells are of great signi cance to functional recovery after SCI.

Discussion
We studied the effects of a natural active peptide on the recovery of spinal cord structure and function after acute SCI.We used behavioral scores, acute-phase biochemical tests, and pathological tests to study SCI recovery.Results showed that OM-LV20 had a positive effect on SCI repair, suggesting that the application of natural active peptides may have broad prospects for the treatment of SCI.
Here, OM-LV20 showed strong ability to promote repair in an acute wound repair model.Whether for skin trauma or acute SCI, several similarities in pathophysiology and repair conditions are found during the acute stage of injury, including activation of in ammatory cells, accumulation of toxic substances, generation of free radicals, and damage to the local microenvironment, which all have important effects on repair after injury 22,23 .In previous reports,intraperitoneal administration of OM-LV20 could signi cantly reduce the infarct area formation, protect cortical and hippocampal neurons from death in I/R rats.Moreover, the underlying molecular mechanism was partly involved with the regulation of the MAPK and BDNF/AKT signaling pathways, as well as TPH1, cAMP, and PAC1R levels 16 .The current research is based on the remarkable ability of OM-LV20 to promote skin injury repair and the neuroprotective effects.
The choice of injury model is important for studies on SCI.At present, the most commonly used models are acute spinal cord contusion, clamping compression injury, ischemic injury, and transected injury 24 .
Here, we chose the complete spinal cord transected injury model 25 .Although this model exhibits high mortality and di culty in nursing, it is one of the most effective models for SCI, and has great value in verifying dynamic changes in the microenvironment at the site of injury and restoration of spinal cord structure and function 26 .
Lipid peroxidation is mediated by free radicals in the acute phase of SCI and has an important effect on secondary injury 27 .As the nal product of lipid peroxidation, MDA levels can indirectly re ect the severity of acute phase cell injury 28 .Similarly, increases in NOx are often observed after injury, and can aggravate tissue damage 29 .Furthermore, both SOD activity 30 and GSH content can re ect the clearance of free radicals after injury 31 .Thus, these bioindicators can be used to judge the severity of SCI during the acute phase of injury.After the occurrence of SCI, the expression levels of BDNF and its receptor TrkB also change signi cantly, and their presence is important for the survival and functional recovery of neuronal cells 32 .BDNF plays a biological role in the activation of full-length TrkB, and an important role in neuronal survival, axon damage, and nerve regeneration 33 .In the spinal cord of normal adult mammals, BDNF is almost undetectable.After SCI, however, BDNF and TrakB are highly expressed in the injured area 34 .In the early stage of injury, BDNF and TrkB are mainly synthesized by neurons and astrocytes; after the acute stage, however, they are mainly synthesized by microglia and macrophages 35 .BDNF and TrkB can help reduce primary injury, protect neuronal cells, and promote axon regeneration 34,36 .At the same time, BDNF can effectively increase the expression of heme oxygenase and heat shock protein in the injured area to reduce secondary injury after SCI.Here, after OM-LV20 treatment of SCI, the content of SOD and GSH in the local spinal cord tissue increased signi cantly, whereas the content of MDA and NOx decreased signi cantly.At the same time, the expression levels of BDNF and TrkB were effectively upregulated, which improved the local microenvironment of the injured site and reduced secondary tissue injury.These favorable environments enhanced neuronal cell survival, with survival status and number of neuronal cells directly determining the degree of functional recovery after injury.
After the acute phase of SCI, a local glial scar gradually develops, which has a decisive role in the recovery of function following injury.After SCI, astrocytes in the damaged area will transform into reactive astrocytes, then mature into scar-like astrocytes, and eventually form a dense glial scar around the injured area.Although the scar tissue in the subacute phase can promote nerve regeneration to a certain extent, scar tissue in the late stage can hinder the regeneration of axons and recovery of spinal cord function, which is a common problem encountered in the treatment of SCI 37 .Scar tissue hinders the axons from crossing the injury site and affects the transmission of biological signals.Thus, the effective removal of old scar tissue should help restore sensory and motor functions after SCI 38 .Although the application of OM-LV20 failed to directly remove old scar tissue, the H&E results (Fig. 3) showed that the active peptide effectively reduced tissue defects at the injury site.More importantly, the old glial scar tissue in the OM-LV20 groups was signi cantly shorter than that in the saline group (Fig. 2), which may be an important reason for the signi cant improvement in hind limb motor function.
In summary, this study showed that the application of an active peptide in the treatment of SCI effectively improved the local microenvironment of the injury, and signi cantly enhanced the structure of the injured spinal cord and function of the hind limbs in rats.These ndings provide strong support for the application of natural active peptides in the treatment of SCI and their use in the development of new drugs and multi-channel combined treatments.

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