hUCMSC-derived Exosomes promote spinal cord repair by alleviating the disruption of the blood-spinal cord barrier

doi:10.21203/rs.3.rs-3511224/v1

Download PDF

Article

hUCMSC-derived Exosomes promote spinal cord repair by alleviating the disruption of the blood-spinal cord barrier

https://doi.org/10.21203/rs.3.rs-3511224/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

After spinal cord injury (SCI), the integrity of the blood–spinal cord barrier (BSCB) is closely related to the occurrence of secondary injury and functional recovery. Previous studies have shown that exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSC-exos) promote nerve repair in multiple degenerative neurological diseases. However, it remains largely elusive whether hUCMSC-exos can promote spinal cord repair after SCI. In this study, hUCMSC-exos were injected through the tail vein immediately after the successful establishment of SCI in a rat model. We showed that the loss of neurons and the destruction of pathological structures were significantly reduced after hUCMSC-exos treatment, and the motor function of rats was also greatly improved. Mechanistically, hUCMSC-exos promote spinal cord repair by affecting autophagy and BSCB integrity. In vitro, the expression of BSCB-associated proteins in human brain microvascular endothelial cells (HBMECs) was significantly reduced in the OGD model, and this reduction was significantly mitigated by treatment with hUCMSC-exos. In addition, autophagic flux was significantly activated after OGD, and hUCMSC-exos treatment further activated autophagic flux. When this process was inhibited by 3-MA (an autophagy inhibitor), the protective effect of hUCMSC-exos was significantly reversed both in vivo and in vitro. These findings suggest that hUCMSC-exos reduce the permeability of the BSCB and promote functional recovery after SCI by activating autophagic flux in endothelial cells. Our study is the first to demonstrate the protective effect of hUCMSC-exos on the BSCB and the underlying mechanism, shedding light on the potential therapeutic benefit for SCI.

Health sciences/Diseases/Trauma

Biological sciences/Stem cells/Mesenchymal stem cells

The barrier function of the blood central nervous system (CNS) across the neuraxial interface consists mainly of the blood‒brain barrier (BBB) and the BSCB, which are highly similar in both morphology and function^{1, 2}. With the help of tight junction (TJ) proteins on the luminal side, highly specialized endothelial cells (ECs) effectively regulate the respiration and energy metabolism of the spinal cord ^{2, 3, 4}. SCI is a catastrophic disease for patients and usually leads to variable degrees of neurological dysfunction, which includes motor, sensory, and autonomic dysfunction ^{5, 6}. Traumatic SCI can be categorized into primary injury and secondary injury in pathophysiology. The initial injury event usually leads to vertebral fractures, spinal cord compression and neuronal damage in the focal area⁷. With the disruption of the BSCB, inflammatory cells, cytokines, and vasoactive peptides enter the damaged area and trigger a sustained secondary injury response, leading to further injury and neurological dysfunction^{2, 7}. Therefore, protecting the BSCB from disruption can effectively improve the prognosis of SCI^{8, 9}.

Autophagy is a conservative lysosomal degradation process that can be used to degrade damaged organelles and misfolded protein polymers to cope with various stresses from both the inside and outside ^{10, 11}. In SCI, the activation of autophagy can effectively reduce endoplasmic reticulum stress and apoptosis, thus providing a protective effect on the BSCB and neurons ⁴. In addition, a recent study showed that autophagy helps to regulate the redistribution of claudin5 across the cell membrane and protects the BBB, which showed a significant increase in permeability after 3-MA inhibition of autophagy ¹². Therefore, growing evidence indicates that autophagy is an important regulator of BSCB protection.

In recent years, exosomes derived from stem cells have been widely used to treat SCI. Compared with mesenchymal stem cells, exosomes are simpler to generate and preserve with few ethical limitations ¹³. Due to the absence of growth and proliferation capacity, exosomes have a lower risk of tumor formation and immune rejection than MSCs ¹⁴. Therefore, exosomes have great potential in the treatment of SCI. A study on Parkinson's disease showed that exosomes secreted by human umbilical cord mesenchymal stem cells (hUCMSCs) could reduce apoptosis of nigrostriatal dopaminergic neurons and increase dopamine levels in the striatum by activating autophagy ¹⁵. In addition, another study showed that hUCMSC-exos could induce M1 to M2 phenotypic polarization and downregulate inflammatory cytokines such as TNF-α, IL-6, IFN-γ and MIP-1α in the treatment of SCI to improve functional recovery ¹⁶. However, it is currently unclear whether hUCMSC-exos have a protective effect on the BSCB after SCI, and the mechanisms involved are still unknown.

In this study, we investigated how hUCMSC-exos regulate autophagic flux to repair BSCB damage after SCI in vivo and in vitro. Through preliminary experiments, we have shown that hUCMSC-exos can reduce the disruption of the BSCB and improve the recovery of spinal cord function by increasing the autophagic flux of endothelial cells after SCI.

2.1 Identification of hUCMSCs and hUCMSC-exos

First, we conducted a thorough characterization of hUCMSCs and exosomes derived from them to verify the successful isolation of hUCMSC-exos. HUCMSCs were grown in a typical mesenchymal stem cell-like spindle shape in vitro under light microscopy (Figure 1A). In addition, flow cytometry analysis showed that these cells expressed high levels of the surface antigens CD73, CD90, and CD105 but were negative for CD34, CD45, and HLA-DR expression, which is consistent with the characteristics of hUCMSCs (Figure 1B). The ability of hUCMSCs to differentiate in multiple directions was also confirmed (Figure 1C). Osteogenic differentiation ability was verified by observing red calcium deposition through Alizarin red staining. Adipogenic differentiation ability was verified by observing a large number of Oil Red O-positive lipid droplets through Oil Red O staining. The chondrogenic differentiation ability was verified by observing the deposition of acidic mucopolysaccharides through Alcian blue staining. The above results indicated that the cells cultured in this study were hUCMSCs that can be used for subsequent experiments. The exosomes secreted by hUCMSCs were separated by ultracentrifugation and then identified. Transmission electron microscopy (TEM) imaging revealed that the morphology of exosomes presented typical cup-shaped spherical vesicles with a bilayer membrane structure (Figure 1D). Furthermore, the results of nanoparticle tracking analysis (NTA) showed that the size distribution of most exosomes varied between 50 and 150 nm (Figure 1E). Western blotting revealed the expression of exosome-specific protein markers, such as CD9, CD63, and TSG101 (Figure 1F). The above results indicate that hUCMSC-exos were successfully isolated. We also investigated the uptake of hUCMSC-exos by HBMECs in vitro. PKH26-labeled exosomes were coincubated with HBMECs for 12 hours and then observed under a confocal fluorescence microscope. The results showed that the exosomes were successfully taken up by the HBMECs and distributed around the nucleus (Figure 1G).

2.2 HUCMSC-exos promote spinal cord repair and motor functional recovery

To investigate the roles of hUCMSC-exos in SCI recovery, rats were injected with hUCMSC-exos via the tail vein immediately after injury for one week, followed by histological correlation assays. Compared to the sham group, rats in the SCI group exhibited significant disruption of spinal cord tissue, including extensive necrosis, vacuole formation, and neuronal loss. Administration of hUCMSC-exos considerably reduced the lesion areas and the loss of Nissl bodies after injury and improved neuronal status (Figure 2A). Additionally, the results of NeuN staining showed similar trends. After SCI, there was a significant reduction in the number of neurons, which was ameliorated by treatment with hUCMSC-exos (Figure 2B and 2C). These results suggested that hUCMSC-exos have therapeutic effects on the injured spinal cord. To further validate the efficacy of hUCMSC-exos in promoting functional recovery after SCI, BBB scores were determined at 1, 3, 5, 7, and 14 days. After SCI, the rats exhibited significant behavioral deficits, indicating that the model was effectively established. From one week after the injury, the SCI+Exos group exhibited a gradual acceleration in motor function improvement compared to the SCI group. In contrast, the score in the sham group remained at 21 throughout, indicating that motor function was unaffected (Figure 2D). These data show that treatment with hUCMSC-exos can effectively improve motor function and provide protective effects on tissues and neurons after SCI.

2.3 HUCMSC-exos attenuate BSCB disruption and prevent the breakdown of TJs and AJs

In recent years, research by Zhang et al. has demonstrated the ability of hUCMSCs to mitigate BBB damage in mice with experimental autoimmune encephalomyelitis ¹⁹. TJs and AJs form the fundamental structure of the BSCB, which plays a significant role in the permeability and functional adjustments of the barrier ^{2, 20}. As accumulating evidence indicates that mesenchymal stem cells impact the central nervous system via paracrine mechanisms, hUCMSC-exos exhibit good penetration of the BBB in a Parkinson's disease rat model and provide protection of the intestinal barrier by decreasing the disruption of TJs in inflammatory bowel disease models^{15, 21}. We propose that hUCMSC-exos promote repair after SCI by protecting the BSCB. To determine whether hUCMSC-exos have a protective effect on the damaged barrier, we evaluated the influence of exosomes on the permeability of the BSCB using Evan's blue assay 24 hours postinjury. The results illustrated that the permeability of the BSCB in the SCI group was increased compared with that in the sham group, while the situation improved after hUCMSC-exos treatment (Figure 3 A and 3B). Next, we examined the protein expression levels of TJs (Claudin 5, occludin, and ZO-1) and AJs (β-catenin) by western blot 3 days after SCI to determine how hUCMSC-exos work. The results showed that the expression levels of Claudin 5, occludin, ZO-1 and β-catenin were decreased to different degrees after SCI. However, after treatment with hUCMSC-exos, the reduced protein expression was recovered significantly (Figure 3 C and 3 D). Double labeling immunofluorescence provides further evidence to support this conclusion. Compared to the sham group, the fluorescence intensity of β-catenin and occludin was greatly reduced after injury in the SCI group. However, after treatment with hUCMSC-exos, this declining trend was substantially alleviated (Figure 3 E-3 H). To further illustrate the protective effect of hUCMSC-exos on the BSCB, we cultured HBMECs in vitro and subjected the cells to oxygen-glucose deprivation. This allowed us to reconstruct the environment experienced by endothelial cells after SCI and analyze the expression of TJs and AJs through western blotting. Consistent with the in vivo results, the expression of Claudin 5, occludin, ZO-1 and β-catenin was decreased greatly to varying degrees in the OGD group compared to the control group. This result was significantly upregulated by hUCMSC-exos (Figure 3I and 3J). In addition, to evaluate the change in the integrity of HBMECs, FITC dextran was added to the cells after OGD. The results showed that compared with the control group, OGD significantly enhanced cell permeability, and this effect was reversed after the addition of exosomes (Figure 3K). The immunofluorescence staining results confirm this conclusion. The intensity of β-catenin and Occludin was decreased in the OGD group compared to the control group, and hUCMSC-exos significantly reversed the destructive effect of OGD (Figure 3L- 3O). These data indicated that hUCMSC-exos prevented the disruption of BSCB integrity and reduced the loss of TJs and AJs after SCI.

2.4 HUCMSC-exos promote autophagic flux via the AMPK/mTOR signaling pathway

Previous research has indicated that the activation of autophagy can protect the integrity of the blood‒brain barrier during I/R injury ²². According to Yang et al., the breakdown of the BBB in secondary damage after stroke is mainly caused by the redistribution of membranous Claudin 5 into the cytosol under hypoxia, which is mediated by caveolin1. By activating autophagy, it can facilitate the degradation of caveolin-1 and aggregate Claudin 5 within the cytosol of BMECs, thus regulating the redistribution of Claudin-5 and reducing injury to the BBB ¹². Previous studies have also shown that hUCMSC-exos can alleviate inflammatory pain and treat Parkinson's disease by inducing autophagy ^{15, 23}. Therefore, we sought to determine whether the protective effects of hUCMSC-exos were related to the activation of autophagic flux and examined autophagy-related proteins in vivo by western blotting. The LC3-II/LC3-I ratio was significantly higher in the SCI group than in the sham group, and this ratio further increased with hUCMSC-exos treatment. The adaptor protein p62 is a substrate of the autophagic process that can reflect the level of autophagic flux, and its accumulation indicates inhibition of autophagic degradation ²⁴.

As shown by western blot, the level of P62 expression increased after SCI, suggesting that autophagic flux is impaired after injury. However, after treatment, the level of P62 was significantly reduced (Figure 4A and 4B). This result indicates that after SCI, autophagic flux is activated, accompanied by disruption. After treatment with hUCMSC-exos, autophagic flux was further activated, and the damage was significantly alleviated. Double-labeling immunofluorescence staining of LC3-II and CD31 showed similar results. In the SCI group, the number of LC3-positive vascular endothelial cells was significantly increased, and this trend was further increased after treatment (Figure 4C and 4D). Cellular experiments also demonstrated that exosomes can effectively promote the activation of autophagic flux and alleviate the impaired autophagic flux caused by OGD (Figure 4E- 4H). In addition, TEM also showed that there were more autophagosomes in the OGD group than in the control group, and this upward trend was more pronounced in the OGD+Exos group, which further supports the above conclusion (Figure 4I). To investigate the potential mechanisms by which hUCMSC-exos regulate autophagic flux, we detected the relevant protein expression of the AMPK/mTOR signaling pathway, which is important for autophagy. The western blot results both in vitro and in vivo showed that hUCMSC-exos significantly increased the p-AMPK/AMPK ratio but decreased the p-mTOR/mTOR ratio (Figure 4J-4 M). These results indicated that hUCMSC-exos could activate autophagic flux via AMPK/mTOR signaling pathways in vitro and in vivo.

2.5 Inhibiting autophagy reverses the protective effect of hUCMSC-exos on BSCB integrity and prevents the loss of TJs and AJs

We next examined whether the barrier protective effect of exosomes is linked to autophagy. After inhibiting autophagy, the results of the Evan's blue assay showed a rise in dye leakage, pointing toward a noteworthy decrease in barrier integrity (Figure 5A and 5B). Then, we used western blotting to measure the expression levels of autophagic flux-related proteins. The ratio of LC3-II/LC3-I expression was significantly lower in the 3MA group than in the group treated only with hucMSCs-exos, while the P62 expression level was significantly higher (Figure 5C and 5D). These results suggest that autophagic flux was effectively inhibited by 3-MA. To determine whether the improvement of TJs and AJs by hUCMSC-exos was dependent on the activation of autophagic flux, we subsequently used Western blotting to detect TJs and AJs. Following the addition of 3-MA to the dosing regimen, the protective effect of hUCMSC-exos on TJs and AJs was significantly decreased (Figure 5E and 5F). To further demonstrate the aforementioned conclusion, we assessed changes in the integrity of HBMECs by adding FITC-dextran after OGD. The results demonstrate a significant increase in cell permeability following the addition of 3-MA (Figure 5G). Detection of cellular proteins related to autophagy via western blot also indicated successful inhibition of autophagic flux through 3-MA in vitro. After the addition of 3-MA, the LC3II/LC3I ratio decreased significantly, while P62 increased significantly compared to that in the hUCMSC-exos group (Figure 5H and 5I). To further verify the protective effects of hUCMSC-exos on the BSCB, we next examined the expression of TJs and AJs in vitro. The results were consistent with those observed in vivo, in which the expression of Claudin 5, occludin, ZO-1, and β-catenin was decreased to varying degrees after the addition of the autophagy inhibitor 3-MA compared with the OGD+Exos group (Figure 5J and 5K). These data demonstrate that the protective effect of hUCMSC-exos on the integrity of the BSCB and the recovery of TJs and AJs depends on the activation of autophagic flux.

2.6 Inhibiting autophagy reverses the protective effect of hUCMSC-exos on functional recovery

To further determine whether hUCMSC-exos have a protective effect on the BSCB and functional recovery via autophagy activation, 3-MA was used to assess exosome efficacy after injury. The HE staining results demonstrated significant tissue structure repair following exosome treatment. However, this protective effect was greatly reversed by the addition of 3-MA. Nissl staining showed similar results. Compared to the group treated solely with exosomes, the morphology and structure of neurons in the SCI+Exos+3-MA group were significantly disrupted, with unclear nucleoli (Figure 6A). The results of NeuN staining provide further confirmation of the previous conclusions. In the SCI+Exos+3-MA group, neuronal morphology and number were drastically reduced as autophagy was inhibited by 3-MA (Figure 6B and 6C). Additionally, the results of the Basso, Beattie, and Bresnahan scoring also demonstrated a noteworthy decrease in rat motor function recovery after the administration of 3-MA. (Figure 6D). These data indicate that the protective effect of hUCMSC-exos on rat motor function and tissue preservation can be reversed by inhibition of the autophagy pathway with 3-MA.

In this study, we demonstrated that hUCMSC-exos have a therapeutic effect on the spinal cord after SCI by preventing destruction of the BSCB and neuronal loss. In this study, we observed that with exosome treatment, the autophagic flux of endothelial cells in the injured spinal cord was significantly activated, thereby providing corresponding protective effects. After inhibition of autophagic flux by 3-MA in vivo and vitro, this therapeutic effect was significantly downregulated. Here, we describe the specific mechanism by which hUCMSC-exos ameliorate BSCB disorder after SCI.

Previous studies have shown that the use of targeted barrier treatments, such as PDGF and bFGF, can effectively alleviate the disruption of the BSCB and promote the recovery of spinal cord function after SCI ^{25, 26}. In recent years, mesenchymal stem cell transplantation has become an emerging treatment for SCI. However, this method also has many problems, such as residual undifferentiated stem cells that may proliferate and form tumors after transplantation, low efficiency of stem cell transformation, and immune rejection ^{27, 28}. Compared with mesenchymal stem cell transplantation, exosomes have the advantages of convenient acquisition, strong targeting, and nontumorigenic properties ¹⁴. Therefore, exosome therapy is gradually becoming a new alternative therapy to mesenchymal stem cell transplantation. A previous study also showed that BMSC-exos can reduce the loss of TJs and AJs in injured endothelial cells through the TIMP2/MMP pathway and significantly reduce the permeability of the BSCB after SCI ⁹. Recently, due to the advantages of ethical access, noninvasive isolation methods, abundant sources, lower immunogenicity and faster self-renewal ability, exosomes derived from human umbilical cord mesenchymal stem cells have been widely used to treat diseases ²⁹. However, no studies have focused on the protective effect of hUCMSC-exos on the BSCB. Therefore, in this study, we used hUCMSC-exos as a treatment for SCI and first validated the protective effect of these exosomes on the BSCB after SCI.

Autophagy is a lysosome-dependent cellular degradation pathway that is critical for the maintenance of cellular homeostasis in the central nervous system ^{30, 31}. The latest research by Yang et al. demonstrates the relationship between autophagic flux and the BBB. By using zebrafish and mouse stroke models, they revealed that the activation of autophagic flux can degrade caveolin 1, as its caveolar endocytosis leads to the loss of claudin5 on the membrane and thus further to a decline in BBB permeability ¹². However, the relationship between the BSCB and autophagy after SCI is still unclear. According to He et al., autophagic flux is disrupted in endothelial cells after SCI, and Kruppel-like Factor 2 contributes to BSCB integrity by augmenting autophagic flux ¹. However, Xu et al. reported opposite results in their study. Although they also observed activation of autophagic flux after SCI, when the rats were treated with edaravone, the expression levels of autophagy and necroptosis-related proteins were significantly decreased, and those of TJs and AJs were increased after treatment. In addition, no accumulation of P62 after injury was observed in their study. As a mediator between LC3 and ubiquitinated substrates, P62 can be degraded in autolysosomes, and its accumulation is often linked to impairment of autophagic flux ³². In our study, we observed that the expression levels of P62 and LC3II were both increased after injury in vivo and in vitro, indicating the disruption of autophagic flux. After treatment with hUCMSC-exos, the level of P62 significantly decreased, while that of LC3II was elevated. This suggests that hUCMSC-exos can promote the activation of autophagic flux. Furthermore, when we used the autophagy inhibitor 3-MA to block autophagic flux, the treatment effect of hUCMSC-exos on BSCB-related proteins was reversed. This indicates that in our study, hUCMSC-exos protected the BSCB by activating autophagic flux. The variation in P62 levels across different studies may be attributed to differences in the severity of injury and in sample collection timing, which can have varying impacts on autophagy activation. Additionally, numerous studies that inhibit autophagic flux to preserve BSCB also result in other types of cell death, making it difficult to distinguish the specific role of autophagic flux in BSCB. Our study demonstrates that hUCMSC-exos have therapeutic effects on SCI and provides evidence supporting the viewpoint that activating autophagic flux can protect the BSCB after injury.

We also tried to explore the upstream mechanism of autophagy. The signaling pathways responsible for autophagy are intricate, with the AMPK/mTOR signaling pathway playing a significant role ³³. AMPK serves as a central sensor for regulating energy homeostasis and can activate autophagy by counteracting mTOR ^{34, 35}. Through western blot analysis, it was determined that treatment with hUCMSC-exos resulted in an increase in the p-AMPK/AMPK ratio and a decrease in the p-mTOR/mTOR ratio. Therefore, we conclude that hUCMSC-exos activate autophagic flux in endothelial cells via the AMPK/mTOR signaling pathways, thereby protecting the damaged BSCB after SCI.

4.1 Cell culture and identification

Human umbilical cord-derived mesenchymal stem cells and stem cell culture medium were purchased from Zhejiang Meisen Cell Technology Co., Ltd. (Zhejiang Province, China). hUCMSCs were cultured in humidified air containing 5% CO_2, and plastic adhesion was observed by light microscopy. The cells were used for experiments at passages 3 and 4. Flow cytometry was used to examine the cell surface markers of hUCMSCs, including CD73, CD90, CD105, CD34, CD45 and HLA-DR (BioLegend, USA). In addition, we used an induced differentiation kit for osteogenesis, adipogenesis and chondrogenesis (OriCell, China) to evaluate the potential multilineage differentiation ability of human umbilical cord-derived mesenchymal stem cells.

Human brain microvascular endothelial cells (HBMECs) were purchased from Shanghai Yuka Biotechnology Co., Ltd. (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) at 37 °C in humidified air with 5% CO₂.

OGD induction was performed in HBMECs to simulate the environment after SCI in vitro. Briefly, the cells were cultured in glucose-deprived DMEM (Gibco, USA) and transferred to a hypoxic chamber with less than 0.5% oxygen. After 6 h of oxygen and glucose deprivation, the HBMECs were transferred to normal conditions for further cultivation for 12 h.

4.2 Isolation, characterization and internalization of exosomes

The 5th generation hUCMSCs were cultured in serum-free medium for 48 h, and then the cell culture medium was collected and centrifuged at 300 × g for 10 min, 2,000 × g for 10 min, and 15,000 × g for 30 min to discard cellular debris. The resulting supernatant was filtered and then ultracentrifuged at 110,000 × g for 70 min (Optima XPN-100, Beckman, United States). The exosome pellet was collected from the bottom and resuspended in phosphate buffer solution (PBS; Gibco). Then, ultracentrifugation was performed again at 110,000 × g for 70 min to remove impurities. The obtained exosomes were stored at -80 °C for subsequent experiments.

Nanoparticle tracking analysis (ZetaView PMX 110, Particle Metrix Meerbusch, Germany) and corresponding software ZetaView 8.04.02 SP2 were used to record the movement video image of exosomes and analyze the particle size distribution. In addition, to further identify exosomes, western blot analysis was used to determine exosome markers, including CD63, CD9 and TSG101 (Abcam, Cambridge, UK).

To observe whether exosomes can be taken up by HBMECs, hUCMSC-exos were labeled with a PKH26 red fluorescent cell inker kit (Sigma Aldrich, USA). According to the manufacturer’s protocol, Diluent C was mixed with PKH26 to prepare the working dye solution and then incubated with exosomes for 10 minutes at room temperature. FBS was added to stop the reaction, and the exosomes were subsequently ultracentrifuged at 100,000 × g for 1 h to remove excess dye. After resuspension, hUCMSC-exos were added to well-grown HBMECs for 12 h of coculture. Fixative and DAPI were added, and the cells were observed and photographed under a confocal microscope.

4.3 Induction of Spinal Cord Injury Rat Model

Female Sprague–Dawley rats were selected for this study because their urethra was shorter than that of male rats and it was easier to urinate artificially after SCI to prevent urinary retention. All healthy adult female Sprague–Dawley rats (average body weight of 220-250 g) were obtained from the animal room of Shanghai Sixth People’s Hospital. All rats were raised under normal conditions with unrestricted access to food and water. All animal studies followed the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. The entire experimental procedure involved was approved by the Animal Research Committee of the Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Before surgery, each rat was anesthetized with 1% (w/v) pentobarbital sodium (50 mg/kg) through intraperitoneal injection. After anesthetization was completed, laminectomy was subsequently performed at the level of the T9-T10 spinal cord. After complete exposure of the dura mater, the rats' spinal cords were subjected to moderate crush injury using the Infinite Horizon Impact Device. The rats in the sham groups received only laminectomy at the same level. After surgery, manual urination was performed three times a day. Antibiotic (cefazolin sodium, 50 mg/kg) was given by intraperitoneal injection once a day.

Rats were randomly divided into the sham group, SCI group, SCI+Exos group and SCI+Exos+3-MA group. The rats in the last three groups were treated with PBS (200 μL), exosomes (200 μg of total exosomal protein in 200 μL of PBS), or 3-MA (2.5 mg/kg in an equivalent volume of PBS) via tail vein injection. The first treatment was given immediately after the injury and then administered once a day for one week.

4.4 Behavioral Tests

After SCI, the degree of functional recovery in rats was evaluated by two independent investigators blinded to the animal groups at 1, 3, 5, 7, and 14 days. As previously described, the Basso, Beattie, and Bresnahan (BBB) scale was used to assess an open field, ranging from 0 to 21 (from complete paralysis to normal hindlimb movement) ^{17, 18}. Before the test, all rats were urinated to ensure that the bladder was empty.

4.5 Western blot analysis

After collecting spinal cord or cell samples, they were placed on ice and homogenized in modified RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). The samples were centrifuged at 14000 g until the supernatant was clear and then collected. The protein concentration was then determined using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Next, proteins were separated by SDS‒PAGE and then transferred to a PVDF membrane (Millipore, USA). After sufficient blocking for 1 h, the membrane was incubated with primary antibody solutions at 4 °C overnight. Then, the membranes were washed with TBS for 10 min three times and incubated with secondary antibodies for 2 h on a shaker. Stripes were visualized and captured by using the ChemiDocTM XRS+Imaging System (Bio-Rad), and then the results were quantified with ImageJ software.

4.6 Transmission electron microscopy

Exosomes were dissolved in 2% paraformaldehyde solution, and then transmission electron microscopy (TEM; H-7500, Hitachi, Tokyo, Japan) was used to observe the morphological characteristics of the exosomes.

To observe the morphology and quantity of autophagosomes, cells were resuspended in PBS, fixed, embedded, and double stained with uranyl acetate/lead citrate. Finally, the prepared sections were observed by transmission electron microscopy (TEM; H-7500, Hitachi, Tokyo, Japan).

4.7 Hematoxylin and eosin staining

After 7 days of SCI, the spinal cord of rats was prepared and fixed in 4% paraformaldehyde in 0.01 M PBS for pathological sectioning of the lesion site. The sections were rinsed in distilled water and then stained with hematoxylin solution for 5 minutes. After dehydration through alcohol, the sections were stained with eosin for 3 min. The prepared sections were then observed and compared.

4.8 Immunofluorescence staining

After fixing with 4% paraformaldehyde, cell and tissue samples were sliced and then incubated with 5% bovine serum albumin for 30 minutes. Subsequently, the sections were incubated with primary antibodies overnight at 4 °C. After incubation, the sections were washed with PBS for 10 minutes three times and then incubated with the appropriate secondary antibody for 1 h at room temperature. Finally, the tissues were treated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes and washed with PBS, and images were captured by using confocal fluorescence microscopy (Nikon, A1 PLUS, Tokyo, Japan).

4.9 Nissl staining

After preparation, the tissue sections were rinsed with distilled water and then incubated with cresyl violet solution for 20 min at room temperature. After dehydration and coverslipping, the slices were observed and photographed for scanning.

4.10 Evans blue dye assays

The permeability of BSCB was examined by Evans blue (EB). One day after injury, rats were injected with 2% Evans blue dye (Solarbio, Beijing, China) through the tail vein. After waiting for 3 h, the EB solution was allowed to circulate fully in the body. The rats were then anesthetized with 1% (w/v) pentobarbital sodium (50 mg/kg) and perfused with normal saline (0.9%). Subsequently, the spinal cord injury site was excised and photographed for recording. After weighing, the tissue was incubated with N,N-dimethyl formamide (Macklin, Shanghai, China) for 3 days at 72 °C. Finally, the supernatant was collected, and the optical density was quantified using a microplate reader (at an excitation wavelength of 620 nm and an emission wavelength of 680 nm).

4.11 Paracellular permeability assay

HBMECs were grown on transwell inserts on a 24-well plate with a 0.4 μm pore size. After receiving OGD for 6 h, fluorescein isothiocyanate (FITC)–dextran (Sigma‒Aldrich, USA) was added to the medium for an additional 2 h of incubation. Subsequently, the fluorescence intensity of FITC-dextran was quantified using a microplate reader (at an excitation wavelength of 493 nm and an emission wavelength of 517 nm).

4.12 Statistical Analysis

In this study, all the data are expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined by Student’s t test when there were only two groups. One-way analysis of variance (ANOVA) and Dunnett's post hoc test were performed when more than two groups needed to be compared. A P value < 0.05 was considered statistically significant.

Conflicts of interest: The authors declare they have no competing interests.

Authors' contributions: Xiaofeng Lian and Jianguang Xu contributed to the study design and the preparation of the manuscript; Zhiheng Chen, Shengting Wu and Yuxuan Qian performed the experiments; Zhiheng Chen and Yuxuan Qian wrote the manuscript; Yinghao Wu, Xin Wang and Fengjie Lu performed data analyses; Erzhu Yang, Guowang Zhang and Baokun Zhang gave constructive advice during the experiments. All authors read and approved the final manuscript.

Ethics approval: All animal experiments were in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health, And the whole procedure involving were approved by the Animal Research Committee of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

Fundings: This work was supported by the National Natural Science Foundation of China (NSFC 81272001)；the Natural Science Foundation of Shanghai of China (23ZR1448100).

Availability of data and material: Research data are stored in an institutional repository and will be shared upon request to the corresponding author.

He Z, Du J, Zhang Y, Xu Y, Huang Q, Zhou Q, et al. Kruppel-like factor 2 contributes to blood-spinal cord barrier integrity and functional recovery from spinal cord injury by augmenting autophagic flux. Theranostics 2023, 13(2): 849-866.
Bartanusz V, Jezova D, Alajajian B, Digicaylioglu M. The blood-spinal cord barrier: morphology and clinical implications. Ann Neurol 2011, 70(2): 194-206.
Mizee MR, de Vries HE. Blood-brain barrier regulation: Environmental cues controlling the onset of barrier properties. Tissue Barriers 2013, 1(5): e26882.
Zhou Y, Zhang H, Zheng B, Ye L, Zhu S, Johnson NR, et al. Retinoic Acid Induced-Autophagic Flux Inhibits ER-Stress Dependent Apoptosis and Prevents Disruption of Blood-Spinal Cord Barrier after Spinal Cord Injury. Int J Biol Sci 2016, 12(1): 87-99.
Badhiwala JH, Wilson JR, Fehlings MG. Global burden of traumatic brain and spinal cord injury. Lancet Neurol 2019, 18(1): 24-25.
Hutson TH, Di Giovanni S. The translational landscape in spinal cord injury: focus on neuroplasticity and regeneration. Nat Rev Neurol 2019, 15(12): 732-745.
Ahuja CS, Wilson JR, Nori S, Kotter MRN, Druschel C, Curt A, et al. Traumatic spinal cord injury. Nat Rev Dis Primers 2017, 3: 17018.
Xin W, Baokun Z, Zhiheng C, Qiang S, Erzhu Y, Jianguang X, et al. Biodegradable bilayer hydrogel membranes loaded with bazedoxifene attenuate blood-spinal cord barrier disruption via the NF-kappaB pathway after acute spinal cord injury. Acta Biomater 2023, 159: 140-155.
Xin W, Qiang S, Jianing D, Jiaming L, Fangqi L, Bin C, et al. Human Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Attenuate Blood-Spinal Cord Barrier Disruption via the TIMP2/MMP Pathway After Acute Spinal Cord Injury. Mol Neurobiol 2021, 58(12): 6490-6504.
Zhang H, Ni W, Yu G, Geng Y, Lou J, Qi J, et al. 3,4-Dimethoxychalcone, a caloric restriction mimetic, enhances TFEB-mediated autophagy and alleviates pyroptosis and necroptosis after spinal cord injury. Theranostics 2023, 13(2): 810-832.
Galluzzi L, Bravo-San Pedro JM, Blomgren K, Kroemer G. Autophagy in acute brain injury. Nat Rev Neurosci 2016, 17(8): 467-484.
Yang Z, Lin P, Chen B, Zhang X, Xiao W, Wu S, et al. Autophagy alleviates hypoxia-induced blood-brain barrier injury via regulation of CLDN5 (claudin 5). Autophagy 2021, 17(10): 3048-3067.
Gimona M, Pachler K, Laner-Plamberger S, Schallmoser K, Rohde E. Manufacturing of Human Extracellular Vesicle-Based Therapeutics for Clinical Use. Int J Mol Sci 2017, 18(6).
Liu WZ, Ma ZJ, Li JR, Kang XW. Mesenchymal stem cell-derived exosomes: therapeutic opportunities and challenges for spinal cord injury. Stem Cell Res Ther 2021, 12(1): 102.
Chen HX, Liang FC, Gu P, Xu BL, Xu HJ, Wang WT, et al. Exosomes derived from mesenchymal stem cells repair a Parkinson's disease model by inducing autophagy. Cell Death Dis 2020, 11(4): 288.
Wang Y, Lai X, Wu D, Liu B, Wang N, Rong L. Umbilical mesenchymal stem cell-derived exosomes facilitate spinal cord functional recovery through the miR-199a-3p/145-5p-mediated NGF/TrkA signaling pathway in rats. Stem Cell Res Ther 2021, 12(1): 117.
Xue MT, Sheng WJ, Song X, Shi YJ, Geng ZJ, Shen L, et al. Atractylenolide III ameliorates spinal cord injury in rats by modulating microglial/macrophage polarization. CNS Neurosci Ther 2022, 28(7): 1059-1071.
Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995, 12(1): 1-21.
Zhang L, Wang X, Lu X, Ma Y, Xin X, Xu X, et al. Tetramethylpyrazine enhanced the therapeutic effects of human umbilical cord mesenchymal stem cells in experimental autoimmune encephalomyelitis mice through Nrf2/HO-1 signaling pathway. Stem Cell Res Ther 2020, 11(1): 186.
Lochhead JJ, Ronaldson PT, Davis TP. Hypoxic Stress and Inflammatory Pain Disrupt Blood-Brain Barrier Tight Junctions: Implications for Drug Delivery to the Central Nervous System. AAPS J 2017, 19(4): 910-920.
Yang S, Liang X, Song J, Li C, Liu A, Luo Y, et al. A novel therapeutic approach for inflammatory bowel disease by exosomes derived from human umbilical cord mesenchymal stem cells to repair intestinal barrier via TSG-6. Stem Cell Res Ther 2021, 12(1): 315.
Li H, Gao A, Feng D, Wang Y, Zhang L, Cui Y, et al. Evaluation of the protective potential of brain microvascular endothelial cell autophagy on blood-brain barrier integrity during experimental cerebral ischemia-reperfusion injury. Transl Stroke Res 2014, 5(5): 618-626.
Hua T, Yang M, Song H, Kong E, Deng M, Li Y, et al. Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis. J Nanobiotechnology 2022, 20(1): 324.
Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 2009, 137(6): 1062-1075.
Ye LX, An NC, Huang P, Li DH, Zheng ZL, Ji H, et al. Exogenous platelet-derived growth factor improves neurovascular unit recovery after spinal cord injury. Neural Regen Res 2021, 16(4): 765-771.
Ye LB, Yu XC, Xia QH, Yang Y, Chen DQ, Wu F, et al. Regulation of Caveolin-1 and Junction Proteins by bFGF Contributes to the Integrity of Blood-Spinal Cord Barrier and Functional Recovery. Neurotherapeutics 2016, 13(4): 844-858.
Sahni V, Kessler JA. Stem cell therapies for spinal cord injury. Nat Rev Neurol 2010, 6(7): 363-372.
Lee AS, Tang C, Rao MS, Weissman IL, Wu JC. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nat Med 2013, 19(8): 998-1004.
Yang W, Zhang J, Xu B, He Y, Liu W, Li J, et al. HucMSC-Derived Exosomes Mitigate the Age-Related Retardation of Fertility in Female Mice. Mol Ther 2020, 28(4): 1200-1213.
Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000, 290(5497): 1717-1721.
Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol 2010, 12(9): 814-822.
Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, Abdellatif M, Abdoli A, Abel S, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)(1). Autophagy 2021, 17(1): 1-382.
Ha J, Kim J. Novel pharmacological modulators of autophagy: an updated patent review (2012-2015). Expert Opin Ther Pat 2016, 26(11): 1273-1289.
Sun L, Zhang S, Yu C, Pan Z, Liu Y, Zhao J, et al. Hydrogen sulfide reduces serum triglyceride by activating liver autophagy via the AMPK-mTOR pathway. Am J Physiol Endocrinol Metab 2015, 309(11): E925-935.
Gu X, Li Y, Chen K, Wang X, Wang Z, Lian H, et al. Exosomes derived from umbilical cord mesenchymal stem cells alleviate viral myocarditis through activating AMPK/mTOR-mediated autophagy flux pathway. J Cell Mol Med 2020, 24(13): 7515-7530.

(Not answered)

Westernblotting.pdf
Original western blots

Download PDF

Version 1

posted

You are reading this latest preprint version

hUCMSC-derived Exosomes promote spinal cord repair by alleviating the disruption of the blood-spinal cord barrier

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results

3. Discussion

4. Materials and methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1