Self-assembled AgNCs with United Umbilical Mesenchymal Stem Cell-Derived Exosome-Repair in Spinal Cord Injury Rats

doi:10.21203/rs.3.rs-3105285/v1

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Self-assembled AgNCs with United Umbilical Mesenchymal Stem Cell-Derived Exosome-Repair in Spinal Cord Injury Rats

https://doi.org/10.21203/rs.3.rs-3105285/v1

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Spinal cord injury (SCI) is a complex process that causes sensory and functional motor deficits, neuronal apoptosis, increased glial scarring, and an inflammatory environment, which is permanent damage. As a therapeutic cell, exosomes can penetrate the blood-brain barrier, regulate inflammation and immune response, inhibit apoptosis, and maintain the blood-spinal cord barrier by promoting angiogenesis and axon outgrowth, promoting the recovery of spinal cord function in rats, is a potential method for the treatment of motor and sensory function in SCI rats, and has attracted much attention in many disease fields. To find a new method for the treatment of spinal cord injury, we choose the Surface enhancement Raman (SERS) and cultured mesenchymal stem cells to obtain exosomes (uMSCEXOs), and then injected them into the injured rats through the tail vein, through observed the damaged parts of the spinal cord of the rats at different time points changes in tissue morphology, changes in inflammatory factors after injury, through the ELISA and SERS detection confirmed the Exos group rats showed high expression of inhibitory inflammatory factor IL-10 and decreased expression of inflammatory factor TNF-α after injury. After damage, the number of neurons in rats treated with exosomes increased. Exosomes will help expand the prospects of umbilical cord mesenchymal stem cells in injury regeneration and repair.

AgNCs

SERS

Spinal Cord Injury

uMSCEXOs

IL-10

TNF-α

SCI as a world medical problem, causes severe limb dysfunction, high disability rate, and many complications^[1]. In the early stage of injury, the inflammatory response plays a vital role in injury, and excessive inflammatory response can cause secondary injury. Neuronal apoptosis, enhanced glial scarring, and an inflammatory environment result from the wounded location^[2]. It can also cause vascular changes, neurons and axons are demyelinated^[3], so repairing damaged nerves and inducing their functional regeneration are the current difficulties in SCI fundamental research ^[4–6].

Exosomes are extracellular vesicles with a diameter in 30 to 200nm, which can penetrate the blood-brain barrier in a small size ^[7–8], exosomes have been found to play an important role in cancer, neurodegeneration and immunity, and containing various functional proteins, mRNAs, microRNAs, and so on^[9–10]. It is regarded as a promising option for cell-free therapeutic uses in repair strategies ^[11–12]. MSC-exosomes play a role in the repair of damaged spinal cord by inducing angiogenesis and axon formation and modulating inflammatory response.

Stem cell transplantation has unique advantages in the treatment of rats with spinal cord injury: umbilical cord stem cells have the characteristics of multi-directional differentiation, low immunogenicity, easy extraction, and long-term survival after transplantation, umbilical cord stem cells have emerged as a novel form of seed cell for current cell transplantation ^[13–14].

The transplantation of neural stem cells into rats can improve the function of their hindlimbs, and the migration of transplanted stem cells and the enrichment of various neurotrophic factors can be detected in the injured part. MSC-derived exosomes have demonstrated to be an appealing cell-free strategy to promoting angiogenesis in various tissue regeneration^[15]. Exosomes derived from bone marrow mesenchymal stem cells are more stable and do not trigger tumorigenesis or immunological rejection. Mesenchymal stem cells produce more exosomes than cells from other sources ^[16–17].

The inflammasome-mediated pyroptosis can be attenuated by modulating BMSC-derived exosomes, exhibit antioxidant effect^[18], lessen oxidative stress and promote nerve function recovery in rats. MiR133b exosomes promote axonal regeneration and motor function recovery after intravenous injection into the tail vein of rats^[19]. We cut the umbilical cord tissue, isolated and cultured mesenchymal stem cells, obtained exosomes (uMSCEXOs), injected them into the injured rats through the tail vein to observe the rats at different time points of spinal cord injury histomorphological changes, and motor function recovery scores. It was found that exosome-injected rats had high expression of IL-10 and low expression of TNF-α. The cyst cavity in the injured area shrunk after 28 days. After fluorescent staining injury, the number of neurons in injured rats increased after the injection of exosomes. Exosome therapy has a repairing effect on spinal cord injury, which will help to expand the prospects of umbilical cord mesenchymal stem cells in injury regeneration and repair in the future.

2.1 Animal preparation and groups

The study used 40 male Sprague-Dawley rats (Shandong First Medical University), weighing 230–250 g. All rats were maintained under a 12 h light/dark cycle pathogen-free condition with free access to food and water. The Shandong First Medical University's Animal Care and Usage Committee gave its approval to the animal use guidelines. Rats were randomized into three groups (n = 5/group): SCI, Exos, and sham groups. The SCI group received SCI induction and were euthanized at days 1, 7, 14 and 28 (n = 5 per time point) for iron content detection. The Sham group rats were intravenously injected with 100 µL of the appropriate exosomes (100 µg/mL) via the tail vein. Rats were administered three total injections, with injections being administered every other day. SCI model rats were instead injected with physiological saline. Rats were then injected with penicillin (2×10⁵ U/kg) once daily for 7 days, and urination care was provided three times per day until the resumption of active urination. Then, 0.1 mL of exosomes were injected into the tail vein for the treatment group.

2.3 Exosome isolation and characterization

An Exo (SBI, USA) was used to collect Umbilical Cord-derived exosomes based upon provided directions. Small bits of umbilical cord tissue was removed. The tissue was centrifuged for 10 min. Then washed by PBS, cultured in DMEM at 37℃ with 5% CO₂ .Using ultracentrifugation technology to separated and purified the exosomes, then verified the formation of Exos by electron microscopy (Fig. 1.) (JEM-1400).

2.4 Rat SCI model development

Rats were intraperitoneally injected with 10% Chloral hydrate, disinfected, and a 2–3 cm incision at the midline of the back was generated centered around the T10 spinous process. Surface musculature was separated to expose the underlying vertebrae, and the T10 spinal cord was fully exposed by removing the spinous processes and lamina. Allen’s striking device was then used to striking force to the T10 spinal cord. After being cleaned with a saline solution containing penicillin, the wound was then stitched up. Successful SCI modeling was confirmed based on rapid blood stasis in the spinal cord and rapid contraction and tremors in the lower limbs of model animals. The sham group rats were anesthetized and had their spinal cords exposed, but no injuring force was applied.

2.5 Neurobehavioral evaluation

Rat locomotor functionality following SCI modeling was assessed based upon Basso-Beattie-Bresnahan (BBB) scores and footprint analysis. Scores were independently determined by two researchers blinded to treatment group assignments and were calculated on 1, 2, 3, 4 weeks post-operation. The footprint analysis to assess physical coordination.

2.6 Enzyme-Linked Immunosorbent Assay (ELISA)

Blood was drawn from the tail vein, and the serum was separated at 3 days after the injury. The levels of TNF-α and IL-10 in the supernatants of lysed cells were determined by using an ELISA kit (Boster, Wuhan, China). OD values at the wavelength of 450 nm were recorded, and the standard curve was used to quantify the expression of TNF-α and IL-10.

2.7 HE

Spinal cord tissue sections were fixed with 4% methyl alcohol, frozen slice and subjected to standard hematoxylin and eosin (H&E) staining. Sections were then dehydrated using ethanol and xylene, sealed using neutral resin, and assessed under an optical microscope.

2.8 Western blot

Tissue protein extraction: 100 mg tissue, shredded with scissors and lysate, homogenized on ice, 5 min by 12000 rpm centrifugation at 4℃, the supernatant was obtained. First, the protein concentration was detected, and the protein was separated by 10% SDS-PAGE and transferred to the nitrocellulose membrane. (Millipore, Jaffrey, NH, USA), NeuN was sealed with 5% skim milk for 1 h, and the first antibody incubated at 4°C overnight. NeuN (1:800, Abcam), GFAP (1:800, Abcam), β-actin (1:2000, Abcam), after cleaning the imprinting, incubating with the second antibody for 1 hour, the protein bands were detected with ECL luminescent kit (Zhongshan Jinqiao, Beijing, China). Image J is used for all density analyses. Immunofluorescence staining.

Sections of spinal cord tissue were washed three times in PBST (10 min/wash) and were then blocked for 1h at room temperature using 5% donkey serum. Sections were then probed with AF647 anti-NeuN (1:400, Abcam) or AF488 anti-GFAP (1:500, Abcam) rats antibodies overnight at room temperature. Sections were then washed thrice with PBS and probed with secondary goat anti-rats IgG (Invitrogen) for 1h. Nuclei were counterstained for 2 min using DAPI, and a nonfluorescent mounting medium (Dako, Glostrup, Denmark) was then used to mount sections to slides prior to laser-scanning confocal microscopy (Olympus). A total of five random fields of view were analyzed per section to assess the numbers of NeuN or GFAP positive cells.

2.9 Statistical analysis

GraphPad Prism v9.0 (GraphPad, San Diego, CA, USA) was used for all statistical analyses. Data are means ± standard deviation (SD) and were compared via t-tests and least significant difference ANOVAs. BBB scores were compared using Mann-Whitney U tests. P < 0.05 was the significance threshold for these studies.

As shown in Fig. 2, AgNCs and GNSs were characterized by scanning electron microscope (SEM), Dynamic Light Scattering (DLS) and ultraviolet-visible-near infrared (UV-vis-NIR). Figure 2a shows the SEM typical of AgNCs, that can be seen as a number of square nanostructures, all being uniform in both size and shape. The particle size of AgNCs was about 60 nm detected by DLS (Fig. 2b). UV-vis-NIR spectra show that the maximum absorption peak is at 550 nm (Fig. 2c). As shown in Fig. 2d-f, GNSs shows many equal size and shape were synthesized,well-defined sharp branches on its surface under SEM. DLS results show that the particle size is about 90 nm, and UV-vis-NIR spectra show that the maximum absorption peak is at 750 nm. In summary, we successfully prepared AgNCs and GNSs providing a detection basis for subsequent experiments.

In order to further verify the positive correlation between IL-10 and TNF-α concentrations in Raman signal intensity, the SERS profiles were diluted to different concentrations (1 fg/mL-1 ng/mL) with PBS and subjected to Raman detection as shown in Fig. 3a and b. As the concentrations of IL-10 and TNF-α increased, the SERS signal intensity also showed a gradual enhanced electromagnetic intensity. We later detected the concentrations of IL-10 and TNF-α in different groups and got Fig. e. Notably, the violin plot revealed that the concentration of TNF-α in the Exo groups were higher than that in the SCI groups compared with the Sham groups, while IL-10 was in the contrary.

Under normal circumstances, IL-10 is expressed at a low level in the endothelial cells of the central canal of the spinal cord, but a certain concentration of IL-10 is not only the basis for maintaining the normal growth and survival of nerve cells, but also affects the differentiation, growth and survival of cells. Figure 3(c, e and f) showed the Exos group rats high expression of inhibitory inflammatory factor IL-10 and decreased expression of inflammatory factor TNF-α after injury. A lot of IL-10 is released locally after injury. To protect the local excessive inflammatory reaction of the injured tissue, it is not difficult to find that the IL-10 of each group begins to increase after injury, initiates the immune cell protection mechanism and participates in the healing of the injured tissue, but compared with the Exos group, the inflammatory factors and glial scar continue to increase in the SCI group, which prevents the regeneration of new neurons and axons in the injured area, and prolongs the inflammatory period.

After the occurrence of SCI, there is ischemia in the injured area, the inflammatory exudate increases, and the glial scar hyperplasia leads to the apoptosis of nerve cells. The regulation of inflammatory response includes the regulation of astrocytes and pro-inflammatory factors ^[20]. In the early stage of injury, vascular rupture leads to edema, upregulation of inflammatory response TNFα, etc., and activates glial cells. Therefore, protecting the damaged area and promoting the growth of blood vessels and neurons is a crucial step in damage repair ^[21].

Postoperative evaluation of the behavior of trunk, tail, and hindlimbs of rats, we injected Exos through the tail vein, compared with the SCI model group, the rats in the exosome treatment group had relatively fewer inflammatory factors in the early stage of injury, and the BBB scores also confirmed that exosomes improved the recovery of hindlimb motor function in injured rats. It is not difficult to find that after 8 weeks of treatment, the motor function of rats in the EXOS group recovered best. The footprint experiment also confirms this. The sham group had a consistent pace and walked freely, the paw support, inter paw coordination, front- and hind paw kinematics, and skilled movements were significantly consistent before and after injury, while the SCI group dragged the hind limbs forward. The Exos-treated rats could perform small floating joint movements, and the hind limb dragging was not obvious in the movement.

Figure 4 shows the HE staining showed that on the 7th day after operation, except for the normal shape of spinal cord in sham group, the nerve fibers in the injured area of SCI group and the Exos group were disordered, extensive nerve cells in white matter degenerated and necrosed, and cysts were formed. In contrast, the Exos group showed less damage and new scar tissue was formed. From the 14th day to the 28th day after operation, the tissue shrank, but the nerve fibers were still disordered and there were obvious cavities. Exos attenuates scar hyperplasia and promotes the recovery of neurological function in rats after injury.

MSCs are multi-lineage differentiated self-renewing pluripotent stem cells ^[22], which can regulate inflammation and immune response, inhibit apoptosis^[23], and maintain the blood-spinal cord barrier by promoting angiogenesis and axon growth integrity, which plays a key role in repairing SCI^[24]. In addition, they can be used to transport genetic material or drugs to target cells, and their relatively small size allows them to penetrate the blood-brain barrier.

The angiogenic effect of umbilical cord Msc-derived exosomes (UCMSCEXOs) in fracture healing, direct injection into rats has therapeutic effects on a variety of orthopedic diseases, and promotes bone repair and regeneration^[25]. After spinal cord injury, the local blood flow decreases, the nerve cells suffer from ischemia and hypoxia, necrosis or apoptosis occurs, and the vascular structure is destroyed. We found through HE staining that the spinal cord tissue in the sham group was intact, while the impact area in the SCI group did not decrease with time. In the exosome treatment group, the cyst cavity was reduced 28 days after surgery.

Bar = 50 µm. (b) Cavities area after the spinal cord injury.

Figure 5. (a) Representative western blotting of Nestin and GFAP with β-actin determined as the internal control. (b) GFAP relative expression (c) Nestin relative expression. Statistical analysis was performed using one-way ANOVA followed Tukey’s multiple comparison test (*p < 0.05).

Figure 6 results showed that the number of GFAP positive cells increased after compared with SCI group, the number of Exos GFAP decreased. The proliferation of Nestin in the peri-injury area was obvious after injury, and the number of Nestin positive cells in the SCI group was higher than that in the SCI group after operation.

Figure 7 shows the results of Nestin, as an intermediate filament protein, is highly expressed in pluripotent stem cells and is a major marker of neural stem cells^[26]. GFAP is a marker of astrocyte activation^[27], increasing in the early stage of injury can protect damaged axons, form glial scar, and avoid secondary injury^[28]. In late stages of injury, extensive glial scar inhibits axonal regeneration^[29–30]. The therapeutic effect of exosomes is not limited to this. We found that the number of neurons in rats treated with exosomes increased after injury. NeuN is a mature neuron-specific nuclear marker protein^[31], and the frequency of NeuN-positive neurons is higher. It shows that exosomes have a repairing effect on the nerve function of rats.

In this study, we were able to determine a facilitative effect on post-injury nerve healing by injecting exosome cells into SCI rats.Through histomorphological changes as well as ELSA inflammatory factor assay and immunofluorescence analysis, we have demonstrated that the exosome groups’ injury area gradually shrank the capsule cavity at 28 days postoperatively and that exosomes underwent changes during their entry into the rats, which not only inhibited inflammation but also improved the post-injury edema state, inhibited glial scar proliferation and promoted neuronal regeneration. Furthermore, we found that exosomes are more successful in restoring motor function in damaged rats and can keep the blood-brain barrier intact. As a result, this work illustrates the critical necessity for the restriction of using exosome therapy alone, we plan to cure spinal cord injury in the future loading exosome on other alternative biomaterials or using it as miRNA carriers in SCI.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Availability of data and materials

not applicable.

Ethical Approval

Approved by Animal Ethics Committee of Shandong First Medical University.

Competing interests

None

Authors' contributions

Qianwen Yue: Methodology, Validation, Data Curation, Formal analysis, Writing-Original Draft. Jixiang Cheng: Data Curation, Validation. Cai Wang: Investigation, Data Curation. Huiyu Wan: Methodology, Resources. Dongyuan Xin: Investigation, Methodology. Ying Wang: Data Curation, Supervision. Jingguo Wu: Validation, Supervision, Writing-Reviewing and Editing. Jingyi Sun: Funding acquisition, Conceptualization,

Funding

This work was supported in part by grants of Natural Science Foundation of Shandong Province (No.ZR2019ZD32, No.ZR2021QH160, No.ZR2021MH0025), Fund of Taishan Scholar Project, Fund of Academic Promotion Program of Shandong First Medical University & Shandong Academy of Medical Sciences (No.2019QL016), and Ji'nan Science and Technology Development Project (202019196).

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Scheme 1 is available in the Supplementary Files section

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Scheme 1. Exosomes genesis and SCI model

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Self-assembled AgNCs with United Umbilical Mesenchymal Stem Cell-Derived Exosome-Repair in Spinal Cord Injury Rats

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Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Animal preparation and groups

2.3 Exosome isolation and characterization

2.4 Rat SCI model development

2.5 Neurobehavioral evaluation

2.6 Enzyme-Linked Immunosorbent Assay (ELISA)

2.7 HE

2.8 Western blot

2.9 Statistical analysis

3. Result and discussion

4. Conclusions

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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