Schwann cell-derived exosomes containing MFG-E8 modify macrophage/microglial polarization for attenuating inflammation via the SOCS3/STAT3 pathway after spinal cord injury methods
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In vivo SCDEs improved functional recovery after SCI
To assess the functional recovery after SCI, the BBB score was observed by two trained observers at 1 day before, and 1, 3, 7, 14, 21, and 28 days after SCI. The BBB score of the SCI + SCDEs group was significantly higher (Fig. ). The results of gait analysis showed that the rear limb coordination of the SCI + SCDEs group was greatly improved, especially in the regularity index, print position, and stance of the hindlimb (Fig. ). Meanwhile, the H&E staining of the bladder showed a lesser thickness of the bladder in the SCI rat treated with SCDEs, which meant that the functions of the bladder recovered faster in the SCDEs treatment group (Fig. ). To assess the improvement in nerve conduction, electrophysiological assay was performed on rats. The MEP results showed that the MEP latency was shortened and the amplitude increased in the SCI + SCDEs group...
Materials and methods
All adult female Wistar rats (the body weight ranging from 200 to 220 g, n = 200) were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China, Permission Number: SCXK (Jing)-2016-0011). All female Wistar rats were housed in the same environments (the temperature ranging from 22 to 24 °C, the humidity ranging from 60 to 80%). All animal experimental protocols were approved by the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (Tianjin, China, Approval Number: IRM-DWLL-2021180). Rats were randomly divided into the following groups: Sham, SCI, SCI + SCDEs, SCI + SCDEs (shMFG-E8-KO), and SCI + SCDEs (shNC). The treatment groups were injected with 250 uL of SCDEs (0.1 ug/uL) transfected with lentivirus, and the SCI group was injected with the same...
Cell culture
Bone marrow was extracted from the femur and tibia of a female Wistar rat. It was incubated in the induction medium containing DMEM/F-12 (Gibco, # 11320033), FBS (10%, Gibco, #10099141 C), penicillin/streptomycin (1%, Gibco, #15070063), and M-CSF (20 ng/ml, #SRP3247, Sigma-Aldrich) at 37 °C in a 5% CO 2 incubator for 7 days. After 7 days of induction, BMDMs could be defined as M0 macrophages and set as the control group, which could be used for subsequent experiments. M0 macrophages were incubated under the condition of LPS (1000 ng/ml, #L3129, Sigma-Aldrich) for 24 h and set as the LPS group. M0 macrophages were incubated under the conditions of LPS and SCDEs (2 µg/ml) for 24 h and set as the SCI + SCDEs group.
The preparation, purification, and identification of SCDEs
When Schwann cells were cultured to the P3-P5, and exosome-free serum was used to prepare the Schwann cell medium. During the cell culture process, the medium was changed every three days. The medium was collected into the centrifuge tube and centrifuged at 4 °C and 300 × g for 5 min to remove dead cells from the medium. Then, the supernatant of the medium was placed at -20 °C for use. After the Schwann cell culture medium was collected, Schwann cell exosomes were obtained through this experiment through ultracentrifugation. In the first step, centrifugation was performed at 4 °C and 2000 × g for 20 min to remove cell residues and organelles, and the supernatant was collected. In the second step, centrifugation was performed at 4 °C and 10000 × g (Centrifuge 5810R, Eppen...
SCDEs uptaken by BMDMs
According to the instructions of the reagent instructions of the PKH26 Red Fluorescent Cell Linker Mini Kit (MINI26-1KT, #MKCM1863, Sigma-Aldrich), SCDEs were incubated with excess dye at room temperature for 4 h and then removed by ultracentrifugation at 100,000 × g for 1 h. After being co-cultured with PKH26-labeled exosomes for 16 h, BMDMs were fixed, penetrated, blocked, and incubated with CD11b antibody (1:400; Abcam, #1211 MA, USA) overnight at 4 °C, and then incubated with secondary antibody. After being washed, the slides were removed from the 24-well plate and mounted with Mounting Medium with DAPI (Abcam, #104139, MA, USA). PKH26-labeled SCDEs being uptaken by primary bone marrow-derived macrophages was observed by the ultra-high resolution laser confocal microscope (ZEISS LSM 900, Germany).
Contusion model of spinal cord injury in rats
The modified Allen method was used to construct the contusion model of SCI in rats, and stability and uniformity of the model were well maintained [ ]. After being weighed, the rats were anesthetized with 5% of isoflurane (RWD lifescience, Shenzhen, China). A dorsal laminectomy was performed on the T10 vertebral body to expose the spinal cord. The impact bar was placed on the spinal cord, and a 10 g node (the diameter of 2.5 mm) was freely dropped from a height of 2.5 cm to create contusion injury with NYU Impactor Model III (W.M. Keck Center for Collaborative Neuroscience, Rutgers, the State University of New Jersey, United States). After hemostasis, the muscle and skin at the incision site is sutured. Successful SCl exhibits the following characteristics in the rats: formation of tail sway reflex, spinal cord ischemia, leg swings, and paralysis [49]. If rats did n...
Hematoxylin and Eosin (H&E) staining
After the paraffin sections were prepared, they were heated in a constant temperature oven at 60 °C for 4 h. Then, the sections were placed in xylene I and II for 10 min and placed in alcohol solutions with different concentrations, including 100%, 95%, 90%, 80%, and 70%, for 5 min each time, and rinsed in distilled water for 1 min. The sections were stained with the hematoxylin (Solarbio, #G1120, Beijing, China) staining solution for 2 min, differentiated with the differentiation solution (1% of hydrochloric acid), washed in distilled water again for 2 min, and then dyed with 0.5% of the eosin staining solution (Solarbio, #G1120, Beijing, China) for 1 min. Finally, in order to complete the dehydration, the tissue sections were successively placed in 70%, 80%, 90%, and 100% gradient ethanol solution. The slides were placed in xylene...
Behavioral evaluation
The BBB functional score was evaluated to assess the recovery of motor function. The score was evaluated at the same time before the surgery, on the day after surgery, on day 3, on day 7, and on the same day every week. The test was performed by two trained researchers (blinded to treatment) to observe and assess the motor function of the rats. The Catwalk-assisted gait analysis (Noldus Information Technology B.V, Netherlands) was performed to evaluate the gait dynamics. On the 28th day after SCDEs treatment, the CatWalk system was used to test the rats in each group. The gait parameters were automatically calculated by the analysis software (CatWalk XT 10.6). This experiment mainly evaluated the effect of the following gait parameters on related behavioral changes after SCI, including regularity index, print position, and stances of the hindlimb. An electrophysiological device (YRKJ-...
Measurement outputs
What raw and processed outputs should exist?
Gait recovery and locomotor function
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- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
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- Group comparisons of gait indices, stride metrics, or recovery curves
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inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
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Evidence quotes (8)
To assess the functional recovery after SCI, the BBB score was observed by two trained observers at 1 day before, and 1, 3, 7, 14, 21, and 28 days after SCI. The BBB score of the SCI + SCDEs group was significantly higher (Fig. ). The results of gait analysis showed that the rear limb coordination of the SCI + SCDEs group was greatly improved, especially in the regularity index, print position, and stance of the hindlimb (Fig. ). Meanwhile, the H&E staining of the bladder showed a lesser thickness of the bladder in the SCI rat treated with SCDEs, which meant that the functions of the bladder recovered faster in the SCDEs treatment group (Fig. ). To assess the improvement in nerve conduction, electrophysiological assay was performed on rats. The MEP results showed that the MEP latency was shortened and the amplitude increased in the SCI + SCDEs group (Fig. ). These results suggested that SCDEs promoted the functional recovery after SCI. Fig. 3 Function recovery of rats by SCDEs after SCI in vivo. A The Flow chart of animal experiments. B, F The BBB scores of Sham, SCI group, and SCI rat group treated with SCDEs ( n = 5). C Represe...
All adult female Wistar rats (the body weight ranging from 200 to 220 g, n = 200) were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China, Permission Number: SCXK (Jing)-2016-0011). All female Wistar rats were housed in the same environments (the temperature ranging from 22 to 24 °C, the humidity ranging from 60 to 80%). All animal experimental protocols were approved by the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (Tianjin, China, Approval Number: IRM-DWLL-2021180). Rats were randomly divided into the following groups: Sham, SCI, SCI + SCDEs, SCI + SCDEs (shMFG-E8-KO), and SCI + SCDEs (shNC). The treatment groups were injected with 250 uL of SCDEs (0.1 ug/uL) transfected with lentivirus, and the SCI group was injected with the same volume of DPBS. Half an hour after the spinal cord injury model was established, SCDEs were injected through the tail vein of rats. We injected rats used in the acute phase (3 days) with SCDEs once a day. Rats in the subacute (2 weeks) and postacute phases (4 weeks) were injected with SCDEs via the...
Bone marrow was extracted from the femur and tibia of a female Wistar rat. It was incubated in the induction medium containing DMEM/F-12 (Gibco, # 11320033), FBS (10%, Gibco, #10099141 C), penicillin/streptomycin (1%, Gibco, #15070063), and M-CSF (20 ng/ml, #SRP3247, Sigma-Aldrich) at 37 °C in a 5% CO 2 incubator for 7 days. After 7 days of induction, BMDMs could be defined as M0 macrophages and set as the control group, which could be used for subsequent experiments. M0 macrophages were incubated under the condition of LPS (1000 ng/ml, #L3129, Sigma-Aldrich) for 24 h and set as the LPS group. M0 macrophages were incubated under the conditions of LPS and SCDEs (2 µg/ml) for 24 h and set as the SCI + SCDEs group.
When Schwann cells were cultured to the P3-P5, and exosome-free serum was used to prepare the Schwann cell medium. During the cell culture process, the medium was changed every three days. The medium was collected into the centrifuge tube and centrifuged at 4 °C and 300 × g for 5 min to remove dead cells from the medium. Then, the supernatant of the medium was placed at -20 °C for use. After the Schwann cell culture medium was collected, Schwann cell exosomes were obtained through this experiment through ultracentrifugation. In the first step, centrifugation was performed at 4 °C and 2000 × g for 20 min to remove cell residues and organelles, and the supernatant was collected. In the second step, centrifugation was performed at 4 °C and 10000 × g (Centrifuge 5810R, Eppendorf, Germany) for 60 min. Subsequently, the collected supernatant was filtered with a 0.22-µm cell filter, and the supernatant was collected for the third time, centrifuged at 130,000 × g and at 4 °C (Beckman Optimal-100 XP, Beckman Coulter, Germany) for 70...
According to the instructions of the reagent instructions of the PKH26 Red Fluorescent Cell Linker Mini Kit (MINI26-1KT, #MKCM1863, Sigma-Aldrich), SCDEs were incubated with excess dye at room temperature for 4 h and then removed by ultracentrifugation at 100,000 × g for 1 h. After being co-cultured with PKH26-labeled exosomes for 16 h, BMDMs were fixed, penetrated, blocked, and incubated with CD11b antibody (1:400; Abcam, #1211 MA, USA) overnight at 4 °C, and then incubated with secondary antibody. After being washed, the slides were removed from the 24-well plate and mounted with Mounting Medium with DAPI (Abcam, #104139, MA, USA). PKH26-labeled SCDEs being uptaken by primary bone marrow-derived macrophages was observed by the ultra-high resolution laser confocal microscope (ZEISS LSM 900, Germany).
The modified Allen method was used to construct the contusion model of SCI in rats, and stability and uniformity of the model were well maintained [ ]. After being weighed, the rats were anesthetized with 5% of isoflurane (RWD lifescience, Shenzhen, China). A dorsal laminectomy was performed on the T10 vertebral body to expose the spinal cord. The impact bar was placed on the spinal cord, and a 10 g node (the diameter of 2.5 mm) was freely dropped from a height of 2.5 cm to create contusion injury with NYU Impactor Model III (W.M. Keck Center for Collaborative Neuroscience, Rutgers, the State University of New Jersey, United States). After hemostasis, the muscle and skin at the incision site is sutured. Successful SCl exhibits the following characteristics in the rats: formation of tail sway reflex, spinal cord ischemia, leg swings, and paralysis [49]. If rats did not show the above symptoms after a single blow and the motor function of their hind limb remained normal on the second day after surgery, they were considered unsuccessful in the construction of a spinal cord injury model and would be excluded from the scope of this study. Within 3 days after SCI, c...
After the paraffin sections were prepared, they were heated in a constant temperature oven at 60 °C for 4 h. Then, the sections were placed in xylene I and II for 10 min and placed in alcohol solutions with different concentrations, including 100%, 95%, 90%, 80%, and 70%, for 5 min each time, and rinsed in distilled water for 1 min. The sections were stained with the hematoxylin (Solarbio, #G1120, Beijing, China) staining solution for 2 min, differentiated with the differentiation solution (1% of hydrochloric acid), washed in distilled water again for 2 min, and then dyed with 0.5% of the eosin staining solution (Solarbio, #G1120, Beijing, China) for 1 min. Finally, in order to complete the dehydration, the tissue sections were successively placed in 70%, 80%, 90%, and 100% gradient ethanol solution. The slides were placed in xylene I and II for 5 min and were sealed with neutral resin (Solarbio, # G8590, Beijing, China). Samples were observed and photographed on a fully automated tissue in situ multi-label landscape quantification analyzer (Vectra Polaris, PerkinElmer).
The BBB functional score was evaluated to assess the recovery of motor function. The score was evaluated at the same time before the surgery, on the day after surgery, on day 3, on day 7, and on the same day every week. The test was performed by two trained researchers (blinded to treatment) to observe and assess the motor function of the rats. The Catwalk-assisted gait analysis (Noldus Information Technology B.V, Netherlands) was performed to evaluate the gait dynamics. On the 28th day after SCDEs treatment, the CatWalk system was used to test the rats in each group. The gait parameters were automatically calculated by the analysis software (CatWalk XT 10.6). This experiment mainly evaluated the effect of the following gait parameters on related behavioral changes after SCI, including regularity index, print position, and stances of the hindlimb. An electrophysiological device (YRKJ-G2008; Zhuhai Yiruikeji Co, Ltd, Guangdong, China) was used to analyze motor evoked potential (MEP) in rats four weeks after SCI to evaluate the recovery of nerve conduction function. The latency and amplitude of MEP were recorded and analyzed in the experiment.
Machine-readable layer
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"datePublished": "2023",
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